bluuebunny/crossref_metadata_2025 huggingface dataset:
Alkaline ribonuclease and ribonuclease inhibitor in mammary gland during the lactation cycle and in the R3230AC mammary tumour
Authors: Liu D K , Williams G H , Fritz P J
Published: August 2015

Alkaline RNAase (ribonuclease) and RNAase inhibitor were assayed to determine the potential role of the degradative process in regulating the amount of RNA in the mammary gland and mammary tumour. Very little free alkaline RNAase activity was found in the cytosol fraction of the mammary gland of virgin, pregnant, lactating or involuting Fischer rats. However, addition of p-chloromercuribenzoate to the assay medium revealed latent RNAase which, when expressed on a DNA basis, decreased during pregnancy and lactation. The cytosol latent RNAase is stable in 0.125 M-H2SO4. The non-cytosol RNAase activity also decreased during pregnancy and lactation. Addition of Triton X-100 produced slightly higher activity at all stages tested. The inhibitor activity in rat mammary gland was very low before pregnancy, increased gradually during pregnancy and more dramatically at parturition, continued to increase throughout lactation and returned to resting-gland values by the sixth day of involution. The increase during pregnancy may be due to the increased cellularity of the gland, whereas the gain during lactation was more than could be accounted for by increases in cell number. The R3230AC transplantable mammary tumour resembles the normal gland in early lactation with respect to both its cytosol and non-cytosol alkaline RNAase activities and its moderately high content of RNAase inhibitor. The relatively high inhibitor and low RNAase activities in both the gland of the lactating rat and in the tumour are of potential significance in maintaining high amounts of RNA and increased rates of protein synthesis in these tissues.
Failure of adrenaline to induce hyperglycaemia after fructose injection in young mice
Authors: Thurston J H , Jones E M , Hauhart R E
Published: August 2015

In control animals a 2-fold increase in liver phosphorylase activity 10min after adrenaline treatment was associated with a 55% increase in plasma glucose (P less than 0.001); at 20 min plasma glucose was 247% of the control value (P less than 0.001). Liver phosphorylase activity was decreased by 74%, 20 min after fructose injection (P less than 0.001), and, although phosphorylase activity increased 5-fold within 5 min of adrenaline injection, no increases in plasma glucose concentration over that found in fructose-injected animals which did not receive adrenaline occurred at either 5, 10 or 20 min. The data confirm inactivation of liver phosphorylase after fructose injection and suggest inhibition of the adrenaline-activated enzyme by the decrease in Pi and elevation of fructose 1-phosphate concentrations produced by the injection of fructose. These findings may be causally related to the hypoglycaemia and the lack of response to glucagon seen in patients with hereditary fructose intolerance after fructose ingestion.
The mannophosphoinositides of Corynebacterium aquaticum
Authors: Hackett J A , Brennan P J
Published: August 2015

Besides the monomannophosphoinositide previously reported in Corynebacterium aquaticum small amounts of other, apparently more glycosylated, mannophosphoinositides have been identified in stationary phase cells. Moreover, by labelling cells with [32P]Pi, phosphatidylinositol was found, comprising about 1.5% of the stationary-phase phospholipids. 2. Pulse-chase experiments performed on cells in the late exponential phase of growth further suggested the sequence phosphatidylinositol leads to monomannophosphoinositide as the first step in the biosynthesis of the mannophosphoinositides. 3. Di-and tri-mannophosphoinositides are apparently the main mannophosphoinositides present during exponential growth. Monomannophosphoinositide predominates only in late stationary phase; in the earlier stationary phase, phosphatidylinositol comprises 50% of the phosphoinositide lipid, and tetramannophosphoinositide constitutes much of the remainder. 4. The metabolism and functions of the mannophosphoinositides are discussed, particularly in relation to changes in their composition throughout the growth cycle.
Specificity of the effect of dietary cholesterol on rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (Short Communication)
Authors: Gregory Keith W. , Booth Roger
Published: August 2015

Dietary cholesterol lowers the activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase without affecting various other liver microsomal enzymes. This is consistent with a specific regulatory mechanism and distinguishes the action of cholesterol on 3-hydroxy-3-methylglutaryl-CoA reductase from that of at least one other stimulus known to affect this enzyme.
A sensitive radiochemical assay for angiotensin-converting enzyme (kinase II)
Authors: Chiu A T , Ryan J W , Ryan U S , Dorer F. E.
Published: August 2015

[[125I]Tyr8]Bradykinin is degraded by angiotensin-converting enzyme to [125I]Tyr-Arg. The reaction product can be separated completely and recovered nearly quantitatively from unchanged substrate by cation-exchange chromatography. Thus it is possible to use [[125I]Tyr8]bradykinin at high specific radioactivity (about 400Ci/mmol) to measure the small quantities of angiotensin-converting enzyme encountered in small-scale cultures of pulmonary endothelial cells.
Acetyl groups in cell-wall preparations from higher plants
Authors: Bacon John S. D. , Gordon Alex H. , Morris E. Jane , Farmer Victor C.
Published: August 2015

O-Acetyl groups were detected by i.r. spectroscopy in cell-wall preparations from grasses and other higher plants and their presence was confirmed chemically. The amounts present are likely to influence both the physical state of the cell-wall polysaccharides and also their digestion by enzymes.
Metabolic regulation of β-galactosidase synthesis in Escherichia coli. A test for constitutive ribosome synthesis
Authors: Dalbow D G , Bremer H
Published: August 2015

The rate of differential synthesis of β-galactosidase (αlac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of αlac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.
Rapid separation of isolated hepatocytes or similar tissue fragments for analysis of cell constituents
Authors: Hems R , Lund P , Krebs H A
Published: August 2015

A simple device is described for the rapid centrifugal separation of isolated hepatocytes or similar tissue fragments from the suspending medium. Rapid separation is essential if meaningful information on the concentrations of cell constituents and their distribution between cell and suspension medium is to be obtained. The usefulness of the technique is illustrated by measurements of hepatocyte constituents (glutamate, aspartate, alanine, K+) which show large concentration gradients against the extracellular medium.
Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver
Authors: Moreno F J , Sánchez-Urrutia L , Medina J M , Sánchez-Medina F , Mayor F
Published: August 2015

1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably < the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.
Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates
Authors: Sugden P H , Newsholme E A
Published: August 2015

1. The activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenases were measured in nervous tissue from different animals in an attempt to provide more information about the citric acid cycle in this tissue. In higher animals the activities of citrate synthase are greater than the sum of activities of the isocitrate dehydrogenases, whereas they are similar in nervous tissues from the lower animals. This suggests that in higher animals the isocitrate dehydrogenase reaction is far-removed from equilibrium. If it is assumed that isocitrate dehydrogenase activities provide an indication of the maximum flux through the citric acid cycle, the maximum glycolytic capacity in nervous tissue is considerably greater than that of the cycle. This suggest that glycolysis can provide energy in excess of the aerobic capacity of the tissue. 2. The activities of glutamate dehydrogenase are high in most nervous tissues and the activities of aspartate aminotransferase are high in all nervous tissue investigated. However, the activities of alanine aminotransferase are low in all tissues except the ganglia of the waterbug and cockroach. In these insect tissues, anaerobic glycolysis may result in the formation of alanine rather than lactate.
The early enhancement of rat liver deoxyribonucleic acid-dependent ribonucleic acid polymerase II activity by tri-iodothyronine
Authors: Jothy S , Bilodeau J L , Champsaur H , Simpkins H
Published: August 2015

It is shown that tri-iodothyronine injected intravenously into thyroidectomized rats induces an early and transient activation of rat liver RNA polymerase II which could be demonstrated to occur 40-80 min after hormonal treatment. There was a simultaneous increase in the concentration of acidic proteins bound to chromatin.
Betaine-induced stimulation of respiration at high osmolarities in a halotolerant bacterium
Authors: Shkedy-Vinkler C , Avi-Dor Y
Published: August 2015

It was shown that non-penetrating solutes at high concentrations inhibit the respiration of the halotolerant bacterium Ba1. Betaine relieved the inhibition caused by osmotic stress and exhibited in this respect a considerable structural specificity. The rate of oxidation of various substrates was stimulated to different extents. It stimulated the rates of both respiration and growth to a similar extent, leaving the energy yield essentially unchanged. In cells pre-loaded with labelled glutamate, betaine also stimulated the rate of oxidation of this intracellular substrate. Betaine was accumulated by respiring cells, and the maximum amount taken up was correlated with the osmolarity of the medium. As judged by chromatography, accumulated intracellular betaine underwent no chemical modification, and this accumulated betaine could not be exchanged with the betaine in the medium or released by passive efflux when respiration was inhibited. Intracellular betaine caused no stimulation of respiration, whereas betaine added to the medium increased the respiratory rate to the same extent in cells pre-loaded with betaine as that in the nonloaded cells. The above observations suggest that iso-economic adjustment is not involved in the anti-osmotic effect of betaine, and that betaine exerts its action on the cellular membrane from the outside.
Stimulation of hepatic mitochondrial calcium transport by elevated plasma insulin concentrations
Authors: Dorman D M , Barritt G J , Bygrave F L
Published: August 2015

The effect of insulin (injected intraperitoneally) on the transport of Ca2+ by hepatic mitochondria from rats was investigated. 2. Elevated concentrations of plasma insulin within the physiological range (10-100muunits/ml) stimulate the initial rate of Ca2+ transport into mitochondria at 4 degrees C by about 75% and prolong by approx. tenfold the time for which the mitochondria retain the accumulated Ca2+. 3. The prolonged retention of Ca2+ is observed under the conditions where hypoglycaemia is significantly decreased by the simultaneous injection of glucose and insulin. 4. A good correlation is observed between the effects on Ca2+ transport and the decrease in blood glucose concentration when the amount of insulin injected was varied. 5. The effects of insulin on mitochondrial Ca2+ transport are apparent at about 30 min after the injection, and are inhibited by cycloheximide. 6. There is little change in mitochondrial energy transduction after the administration of insulin. 7. The results are briefly discussed in relation to the mechanisms of Ca2+ transport across the inner mitochondrial membrane and the role of mitochondria in modifying intracellular Ca2+ concentrations with reference to the mechanism(s) by which insulin affects cellular metabolism.
Ribosome function in livers of porphyric mice
Authors: Del Favero A , Gamulin S , Gray C H , Norman M R
Published: August 2015

1. A porphyrinogenic drug, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, caused a decrease in the proportion of single (‘run off’) ribosomes, and an increase in the number of polyribosomes, in the livers of treated animals. 2. No change could be detected in the distribution of amino acid incorporation among hepatic polyribosomes.
Immunochemical relationship between α-amylases of rat liver, serum, pancreas and parotid gland
Authors: Messer M , Dean R T
Published: August 2015

1. Rabbit antisera to purified rat parotid α-amylase were prepared. 2. The relationships between rat parotid-gland, pancreatic, serum and liver amylase were investigated by using the antisera in immunodiffusion, immunoelectrophoresis and immunoinhibition experiments. 3. Serum and liver amylase were identical, and very similar to parotid-gland amylase; pancreatic amylase was, however, quite distinct, and showed only some of the determinants present on parotid-gland amylase. 4. The data strengthen the suggestion that the liver is the main source of serum amylase.
The mechanism of polyribosome disaggregation in brain tissue by phenylalanine
Authors: Taub F , Johnson T C
Published: August 2015

The injection of neonatal mice with phenylalanine resulted in a rapid decrease in brain polyribosomes and a concomitant increase in monomeric ribosomes. Animals of 1-16 days of age were equally affected by phenylalanine, although the brain polyribosomes of 60-day-old mice were relatively resistant to the effects of phenylalanine. The population of free polyribosomes appeared to be more sensitive to phenylalanine treatment than bound polyribosomes, which were somewhat more resistant to disruption by high concentrations of the amino acid. The effects of phenylalanine were more pronounced with polyribosomes in the cerebral cortex than with those in the cerebellar tissue. The mechanism of polyribosome disruption was shown to be independent of hydrolysis mediated by ribonuclease. Virtually all of the monomeric ribosomes that resulted from phenylalanine treatment were shown to be inactive with regard to endogenous protein synthesis and were present in the cell cytoplasm as vacant couples. These ribosomes were readily dissociated by treatment with 0.5 M-KCl and subsequent ultracentrifugation. These results are discussed in the light of the possibility that high concentrations of phenylalanine disrupt brain protein synthesis by a molecular mechanism that is associated with initiation events.
Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor-bean endosperm
Authors: Lord J M
Published: August 2015

Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.
Characterization by gas-liquid chromatography-mass spectrometry and proton-magnetic-resonance spectroscopy of pertrimethylsilyl methyl glycosides obtained in the methanolysis of glycoproteins and glycopeptides
Authors: Kamerling J P , Gerwig G J , Vliegenthart J F G , Clamp J R
Published: August 2015

The quantitative analysis by gas chromatography of monosaccharides present in glycoproteins and glycopeptides using methanolysis, followed by re-N-acetylation and trimethylsilylation, gives rise to several peaks for each monosaccharide. The identity of these peaks for xylose, fucose, mannose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid was established for α- and β-methyl pyranosides and furanosides by combined g.l.c.-mass spectrometry and proton-magnetic-resonance spectroscopy. These data provide for the unambiguous interpretation of the gas chromatograms obtained in the application of this g.l.c. method, and supply basic information for the further application of mass spectrometry in this field.
The polypeptide composition of bovine epidermal α-keratin
Authors: Steinert P M , Idler W W
Published: August 2015

1. The polypedtide chains that comprise the subunits of the tonofilaments, or the α-keratin component, of bovine epidermis were fractionated by combination of chromatography on DEAE-cellulose and preparative polyacrylamide-gel electrophoresis. 2. The seve polypeptide chains investigated had generalyy similar properties; all contained two residues per molecule of tryptophan and N-acetylserine was the common N-terminal amino acid residue. 3. On the basis of close similarities in α-helix content and amino acid composition, the polypeptide chains were classified into three distinct groups. Each group contained approximately one-third of the total polypeptides on a molar basis. The groups and designated polypeptides chain numbers were: group one, polypeptides 1a and 1b, which had moleculae weights of 58,000, contained about 25% α-helix, 86 glutamic acid and 8 cysteine residues per molecule, but which differed in net charge, extinction coefficients and tyrosine contents; group two, polypeptides 2, 3, and 4, which hadmolecular weights within thewithin the range of 52,00-56,000, contained about 48% α-helix, 54 glutamic acid and 6 cysteine residues per molecule, but which differed in extinction coefficients and tryosine contents; and group, polypeptides 5 and 6, which had molecular weights of 47000-48000, contained about 56% α-helix, 64 glutamic acid and 4 cysteine residues per molecule, but which differed in extinction coefficients and tyrosine contents, it is suggested that none of the chains is a precursor or a degradation product of other polypeptidc chains. 5. It is concluded that bovine epidermal α-keratin consists of a heterogeneous group of similar polypeptide chains.
The action of hydrogen peroxide on the hydroxylation of p-coumaric acid by spinach-beet phenolase
Authors: Vaughan P F T , McIntyre R J
Published: August 2015

Treatment of spinach-beet phenolase with H2O2 under aerobic conditions results in a stimulation of the p-coumaric acid hydroxylation it catalyses, but not the caffeic acid oxidation. Spectroscopic evidence suggests that an oxygenated enzyme species is formed under these conditions.
Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland
Authors: Auricchio Ferdinando , Satriano Rocco , Rotondi Andrea , Quirino Ada , Bresciani Francesco
Published: August 2015

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [3H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[3H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17β into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [3H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [3H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [3H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15°C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by α-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17β in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17β injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [3H]uridine uptake into RNA are not precursor-pool-dependent.
Reconstitution of pig lymphocyte plasma membranes from solubilized components, with particular reference to membrane-associated immunoglobulins
Authors: Chavin Stephen I. , Holliman Andrew
Published: August 2015

Plasma membranes of lymphocytes obtained from pig mesenteric lymph nodes were reconstituted after solubilization with bile salts. The proportion by weight of immunoglobulin in the reconstituted membrane was no greater than about 5–10% of that in the original membrane. Possible reasons for the low reincorporation of immunoglobulin are discussed.
Hormonal control of Luteal 20α-Hydroxy steroid dehydrogenase and Δ5-3β-hydroxy steroid dehydrogenase during luteolysis in the pregnant rat
Authors: Rodway R G , Kuhn N J
Published: August 2015

Treatment of pregnant rats with human chorionic gonadotrophin, luteotrophin (luteinizing hormone), luteotrophin-releasing hormone, prostaglandin F2α, aminoglutethimide, or by foetoplacental removal or hysterectomy achieved a common multiple-response pattern, namely increased activity of luteal 20α-hydroxy steroid dehydrogenase with decreased activity of delta5-3β-hydroxy steriod dehydrogenase and release of delta4-3-oxo steroids in vitro. 2. Similar effects of foetoplacental removal are noted in pregnant mice. 3. Gonadotrophin induced lower activities of 20α-hydroxy steroid dehydrogenase, except at the very end of pregnancy, and partly inhibited the induction caused by foetoplacental removal. 4. The results suggest that existence of a placental factor that restrains these changes until the end of normal pregnancy, which is produced in amounts proportional to the number of placentae and is conveyed to the ovary via the blood. 5. This factor was not replaced by prolactin. 6. It is argued that neither placental lactogen nor pituitary luteotrophin participate in the induction of 20α-hydroxy steroid dehydrogenase at late pregnancy in the rat. 7. Aminoglutethimide induced 20α-hydroxy steroid dehydrogenase only in late pregnancy. This was partly reversed by progesterone, wholly reversed by progesterone plus oestrogen, and did not involve the pituitary.
Evidence against the participation of the γ-glutamyltransferase-γ-glutamylcylclotransferase pathway in amino acid transport by rabbit erythrocytes
Authors: Young J D , Ellory J C , Wright P C
Published: August 2015

The GSH concentration of rabbit erythrocytes was monitored under conditions of large net transport of alanine, phenylalane and lysine in the absence of glucose. In no case was there an appreciable alteration in GSH concentration during amino acid uptake. It is suggested that the γ-glutamyltransferase-γ-glutamylcyclotransferase pathway does not participate in amino acid transport by these cells.
A phospholipid-deacylating system of bacteria active in a frozen medium
Authors: Hazlewood G P , Dawson R M C
Published: August 2015

A phosphatidylcholine-deacylating system present in a Butyrivibrio species (probably fibrisolvens) shows appreciable activity at low temperatures with a maximum hydrolysis rate at—10 degrees C. 2. The rate at—10 degrees C is higher than at 39 degrees C unless the system at the latter temperature is stimulated by adding oleic acid or sodium dodecyl sulphate. 3. The low-temperature phospholipase activity has an absolute requirement for thiol reagents, e.g. cysteine, dithiothreitol or mercaptoethanol. 4. Ca2+, Mg2+ and Mn2+ stimulate the activity up to 10 mM, but EDTA inhibits; higher concentrations of Ca2+ also inhibit. 5. The enhancement of activity at low temperatures appears not to be associated with a crystalline change in the hydrated phospholipid substrate, but depends on the formation of a solid phase in the incubation medium which brings the substrate and bacterial cells into juxtaposition or causes fusion.
Kinetics of Alkaline Phosphatase from Pig Kidney. Influence of complexing agents on stability and activity
Authors: Ackermann B P , Ahlers J
Published: August 2015

Metal ion-complexing agents, like KCN, EDTA etc., inactivate alkaline phosphatase of pig kidney. This inactivation is reversible at low concentrations of the complexing agents and irreversible at high concentrations. The reversible inhibition is probably due to removal of Zn2+ ions from the active site, where they are necessary for catalytic action, whereas the irreversible inhibition results from the removal of Zn2+ ions necessary for preservation of the structure. The inactivation is pseudo-first order. It depends on the concentration, size and charge of the complexing agents. β-Glycerophosphate and Mg2+ ions protect the enzyme from inactivation by complexing agents. Quantitative examination of the effect of substrate leads to a model that is similar to the “sequential model” proposed by D.E. Koshland, G. Nemethy & D. Filmer (1966) (Biochemistry 5, 365-385) to explain allosteric behavior of enzymes. It describes the sequential addition of two substrate molecules at two active centres of the dimer enzyme. The binding of the substrate molecules is accompanied by changes in the conformation, which lead to stabilization of the enzyme against attack by complexing agents.
Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen
Authors: Lee-Own V , Anderson J C
Published: August 2015

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.
Chiral recognition of prochiral centres and general acid-base catalysis. Necessarily interrelated manifestations of active-site structure
Authors: Südi J
Published: August 2015

On the basis of Ogston's [(1948) Nature (London) 162, 963] argument, the following conclusions are indicated by the stereochemistry of the reversible oxidation of glycollate by lactate dehydrogenase: (1) general acid-base catalysis is involved in the reaction; (2) the transformation of enzyme-bound (tetrahedral) substrate into enzyme-bound (trigonal) product involves a conformational transition of the enzyme-coenzyme complex.
Electron transfer between soluble and immobilized mammalian cytochrome c. Equilibrium and kinetic studies on immobilized cytochrome c
Authors: Colosimo A , Brunori M , Antonini E
Published: August 2015

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.
Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin
Authors: Wang T T , Hofmann T
Published: August 2015

A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.
The effect of detergents on bovine adrenal ferredoxin
Authors: Mardanyan S S , Grigoryan N A , Nalbandyan R M
Published: August 2015

The incubation of adrenal ferredoxin with various detergents in the presence of oxygen or ferricyanide leads to bleaching of the protein. The bleached preparation has the properties of apoferredoxin and it can be reconstituted with high yield by conventional methods.
The regulation of haemoglobin synthesis in cultured chick blastoderms by steroids related to 5β-androstane
Authors: Irving R A , Mainwaring W I P , Spooner P M
Published: August 2015

1. After 24h of incubation, the blastoderm may be dissected from the early developing chick embryo and successfully maintained under conditions of organ culture in vitro. 2. Low concentrations of steroids related to 5beta-androstane stimulate the synthesis of foetal haemoglobins, types E and P, in a highly steroid- and tissue-specific manner.
The metabolism of the phosphonium analogue of choline in cultured cells. A useful nuclear-magnetic-resonance probe for membrane phosphatidylcholine
Authors: Sim E , Pasternak C A
Published: August 2015

1. Replacement of choline by the phosphonium analogue does not affect the growth rate of P815Y, NIL, 3T3, and SV40/3T3 cells in culture. 2. The fatty acid composition of the resulting phosphonium phosphatidylcholine is similar to that of phosphatidylcholine. 3. The rate of synthesis and degradation of phosphatidylcholine and of the phosphonium analogue are similar. 4. Phospholipid-exchange protein does not distinguish between phosphatidylcholine and the phosphonium analogue. 5. By contrast, incorporation of phosphonium choline into sphingomyelin occurs to only a minor extent. 6. It is concluded that, since the enzymes involved in the turnover of phosphatidylcholine do not discriminate between quaternary N and quaternary P in the polar head-group region, phosphonium choline should prove to be a useful probe for 31P nuclear-magnetic-resonance (n.m.r.) studies of natural membranes.
The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria
Authors: Ragan C. I
Published: August 2015

Preparations of NADH-ubiquinone reductase from bovine heart mitochondria (Complex I) were shown to contain at least 16 polypeptides by gel electrophoresis in the presence of sodium dodecyl sulphate. 2. High-molecular-weight soluble NADH dehydrogenase prepared from Triton X-100 extracts of submitochondrial particles [Baugh & King (1972) Biochem. Biophys. Res. Commun. 49, 1165-1173] was similar to Complex I in its polypeptide composition. 3. Solubilization of Complex I by phospholipase A treatment and subsequent sucrose-density-gradient centrifugation did not alter the polypeptide composition. 4. Lysophosphatidylcholine treatment of Complex I caused some selective solubilization of a polypeptide of mol.wt. 33000 previosuly postulated to be the transmembrane component of Complex I in the mitochondrial membrane [Ragan (1975) in Energy Transducing Membranes: Structure, Function and Reconstitution (Bennun, Bacila & Najjar, eds.), Junk, The Hague, in the press]. 5. Chaotropic resolution of Complex I caused solubilization of polypeptides of molecular weights 75000, 53000, 29000, 26000 and 15500 and traces of others in the 10000-20000-mol.wt.range. 6. The major components of the iron-protein fraction from chaotropic resolution had molecular weights of 75000, 53000 and 29000, whereas the flavoprotein contained polypeptides of molecular weights 53000 and 26000 in a 1:1 molar ratio. 7. Iodination of Complex I by lactoperoxidase indicated that the water-soluble polypeptides released by chaotropic resolution, in particular those of the flavoprotein fraction, were largely buried in the intact Complex. 8. The polypeptides of molecular weights 75000, 53000, 42000, 39000, 33000, 29000 and 26000 were present in 1:2:1:1:1:1:1 molar proportions. The two subunits of molecular weight 53000 are probably non-identical.
Identification of bile acids in the serum and urine in cholestasis. Evidence for 6α-hydroxylation of bile acids in man
Authors: Summerfield J A. , Billing B H. , Shackleton C H. L.
Published: August 2015

In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3β, 7α-dihydroxy-5β-cholan-24-oic acid, 3α, 6α-dihydroxy-5β-cholan-24-oic acid (hyodeoxycholic acid), 3α, 6α, 7α-trihydroxy-5β-cholan-24-oic acid (hyocholic acid) and 3α, 7β, 12α-trihydroxy-5β-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 β-cholestane-3α, 7α, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations.
Effects of calcium ions and adenosine diphosphate on the activities of NAD+-linked isocitrate dehydrogenase from the radular muscles of the whelk and flight muscles of insects
Authors: Zammit V A. , Newsholme E A.
Published: August 2015

1. The activity of NAD+-linked isocitrate dehydrogenase from the radular muscle of the whelk is higher than those in many vertebrate muscles and only slightly lower than in the flight muscles of insects. The enzyme activity from the whelk (Buccinum undatum) is stable for several hours after homogenization of the radular muscle, whereas that from insect flight muscle is very unstable. Consequently, the enzyme from the whelk muscle is suitable for a systematic investigation of the effects of Ca2+ and ADP. 2. The sigmoid response of the enzyme activity to isocitrate concentration is markedly increased by raising the Ca2+ concentration from 0.001 to 10 muM, but it is decreased by ADP. The inhibitory effect of Ca2+ is most pronounced at pH7.1; it is not observed at pH 6.5. Similar effects are observed for the enzyme from the flight muscle of the locust (Schistocerca gregaria) and the water bug (Lethocerus cordofanus). The percentage activation by ADP of the enzyme from either the whelk or the insects is greater at 10 muM-Ca2+, and 50% of the maximum activation is obtained at 0.10 and 0.16 mM-ADP for the enzyme from whelk and locust respectively at this Ca2+ concentration. At 10 muM-Ca2+ in the absence of added ADP, the apparent Km for isocitrate is markedly higher than in other conditions. Ca2+ concentrations of 0.01, 0.1 and 0.2 muM cause 50% inhibition of maximum activity of the enzyme from the muscles of the whelk, locust and water bug respectively. 3. Recent work has indicated that mitochondria may play a complementary role to the sarcoplasmic reticulum in the control of the distribution of Ca2+ in muscle. The opposite effects of Ca2+ on the activities of isocitrate dehydrogenase and mitochondrial glycerol phosphate dehydrogenase from muscle tissue are consistent with the hypothesis that changes in the intracellular distribution of Ca2+ control the activities of these two enzymes in order to stimulate energy production for the contraction process in the muscle. Although both enzymes are mitochondrial, glycerol phosphate dehydrogenase resides on the outer surface of the inner membrane and responds to sarcoplasmic changes in Ca2+ concentration (i.e. an increase during contraction), whereas the isocitrate dehydrogenase resides in the matrix of the mitochondria and responds to intramitochondrial concentrations of Ca2+ (i.e. a decrease during contraction). It is suggested that changes in intramitochondrial Ca2+ concentrations are primarily responsible for regulation of the activity of NAD+-isocitrate dehydrogenase in order to control energy formation for the contractile process. However, when the muscle is at rest, changes in intramitochondrial concentrations of ADP may regulate energy formation for non-contractile processes.
Effect on uridine diphosphate glucuronyltransferase activity of depleting and restoring phospholipids to guinea-pig liver microsomal preparations
Authors: Berry C S. , Caldecourt M , Hallinan T
Published: August 2015

Phospholipid depletion substantially inhibited the maximum demonstrable activities of the forward (glucuronidation) and reverse reactions of UDP-glucuronyltransferase towards p-nitrophenol in guinea-pig liver microsomal preparations. Dispersions of liver phospholipids restored activity, whereas non-phospholipid amphipaths failed to do so effectively. These results suggest that the system is probably phospholipid-dependent rather than conformationally constrained by phospholipids.
Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement
Authors: Reid K B M
Published: August 2015

1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments.
Purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity
Authors: Allen A K , Desai N N , Neuberger A
Published: August 2015

1. The lectin from the broad bean (Vicia faba) was purified by affinity chromatography by using 3-O-methylglucosamine covalently attached through the amino group to CH-Sepharose (an omega-hexanoic acid derivative of agarose). Its composition and the nature of its subunits were compared with concanavalin A and the lectins from pea and lentil. 2. Unlike the other three lectins, broad-bean lectin is a glycoprotein; a glycopeptide containing glucosamine and mannose was isolated from a proteolytic digest. 3. The mol.wt. is about 47500; the glycoprotein consists of two apprently identical subunits, held together by non-covalent forces. Fragments of the subunits, similar to those found in concanavalin A and soya-bean agglutinin, were found in active preparations. 4. Broad-bean lectin was compared with concanavalin A and the lectins from pea and lentil in an investigation of the inhibition of their action by a number of monosaccharides, methyl ethers of monosaccharides, disaccharides and glycopeptides. The most striking differences concern 3-O-substituted monosaccharides, which are strong inhibitors of the action of broad-bean, pea and lentil lectins but not of the action of concanavalin A. There is, however, no strong inhibition of the action of these lectins by 3-Olinked disaccharides.
State and accessibility of zinic in yeast alcohol dehydrogenase
Authors: Leskovac V , Trivić S , Latkovska M
Published: August 2015

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited in the presence of 1,10-phenanthroline. 2. A conformational change in the enzyme's structure is induced by 1,10-phenanthroline, and is abolished in the presence of NADH. 1,10-Phenanthroline binds to the enzyme competitively with respect to NADH, with a stoicheiometry of 2 mol of 1,10-phenanthroline/144000g of enzyme. 3. 1,10-Phenanthroline induces a time-dependent dissociation of Zn2+ from the enzyme, which is in correlation with its inhibitions. 4. Spectrophotometric measurement indicates that the dissociation of half (2 zinc atoms/tetramer) of the total zinc content of the enzyme correlates with the full inhibition of its activity. Measurement of the tightly bound Zn2+ by atomic absorption photometry confirms this. 5. A proposition is advanced that the tetrameric molecule of yeast alcohol dehydrogenase possesses an inherent asymmetry, with four monomeric subunits being arranged in two mutually symmetrical pairs.
The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation
Authors: Robson B , Pain R H
Published: August 2015

1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with ‘native format’ [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.