bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of gluconeogenesis in isolated rat liver cells by methylenecyclopropylpyruvate (ketohypoglycin)
Authors: Kean E A , Pogson C I
Published: August 2015

1. In isolated rat liver cells, hypoglycin is a less effective inhibitor of gluconeogenesis than its transamination product, methylenecyclopropylpyruvate (ketohypoglycin). 2. Methylenecyclopropylpyruvate at 0.3 mM inhibits gluconeogenesis from all substrates tested, except fructose. 3. Methylenecyclopropylpyruvate does not affect 14CO2 release from [1(-14)C]palmitate, but, in the absence of lactate, inhibits ketogenesis and causes a decrease in the [beta-hydroxybutyrate]/[acetoacetate] ratio. These effects are masked when lactate (10 mM) is present. 4. In the presence of lactate and palmitate, 0.3 mM-methylenecyclopropylpyruvate produces a fall in total acid-soluble CoA and a relative increase in short-chain acyl-CoA at the expense of CoA and acetyl-CoA without changing the ATP, ADP and aspartate contents or the [lactate]/[pyruvate] ratio. 5. Many of the effects of methylenecyclopropylpyruvate observed are consistent with inhibition of butyryl-CoA dehydrogenase and of specific CoA-dependent enzymes involved in gluconeogenesis.
N-formyl-l-methionine deformylase activity in human leucocytes and platelets
Authors: Ackerman S K , Douglas S D
Published: August 2015

Extracts of human neutrophils, lymphocytes and platelets enzymically deformylate N-formyl-L-methionine. Enzyme activity is stimulated by Co2+, inhibited by bivalent-cation chelators and unaffected by inhibitors of serine, thiol and carboxyl proteinases. Leucocyte or platelet N-formylmethionine deformylase may be important in modulation of neutrophil responses to chemoattractant formylmethionyl peptides or similar compounds.
Isolation of material displaying insulin-like immunological biological activity from the brain of the blowfly Calliphora vomitoria
Authors: Duve H , Thorpe A , Lazarus N R
Published: August 2015

An insulin-like material from the brain of the blowfly Calliphora vomitoria was partially purified by acid alcohol extraction, gel filtration and ion-exchange cellulose chromatography. In addition, the RF value on polyacrylamide-gel electrophoresis was determined. The material was characterized by its ability to cross-react with bovine insulin antibody and by displaying diminished immunoreactivity on dilution. It displaced specifically bound 125I-labelled insulin from rat liver plasma membrane insulin receptors and displayed insulin-like biological activity on the isolated rat fat-cell. Within 30 min of injection into Calliphora, made hypertrehalocaemic and hyperglucaemic as a result of median neurosecretory cell removal, it caused the concentrations of both sugars to return to normal. The hypothesis is put forward that the median neurosecretory cells are the source of the material.
Inhibition of glutamine synthetase activity by manganous ions in a cytosol extract of rat liver
Authors: Joseph S K , Bradford N M , McGivan J D
Published: August 2015

Glutamine synthetase activity in a cytosol extract of liver was inhibited non-competitively by Mn2+ ions. The apparent Ki for Mn2” in the presence of phosphate was 8 micro M. Inhibition of glutamine synthetase by intracellular Mn2+ may contribute to the very low rates of glutamine synthesis observed in perfused liver and isolated hepatocytes.
Hydrolysis of an exogenous 125I-labelled protein by rat yolk sacs. Evidence for intracellular degradation within lysosomes
Authors: Livesey G , Williams K E
Published: August 2015

When added to the serum-free medium in which 17.5-day rat yolk sacs were incubated, formaldehyde-denatured 125I-labelled bovine serum albumin was rapidly degraded. More than 80% of the radiolabelled digestion products appearing in the incubation medium consisted of [125I]iodo-L-tyrosine; larger digestion products were found only in association with the yolk-sac tissue. In the early stages of an incubation, low-molecular-weight digestion products began to appear in the incubation medium only after they could be detected within the tissue, and progressive association of trichloroacetic acid-insoluble radioactivity with the tissue preceded both these events. None of the observed proteolysis could be attributed to proteinases released into the incubation medium. Tissue-associated acid-insoluble radioactivity showed a lysosomal distribution on sub-cellular fractionation, and cell-free homogenates of yolk sacs degraded albumin only at acid pH values. Progressively decreasing the rat of pinosome formation (either by progressively lowering the incubation temperature or by the use of increasing concentrations of the metabolic inhibitor rotenone) caused a corresponding decrease in the rate of degradation of albumin. These findings indicate that, in vitro, formaldehyde-denatured 125I-labelled bovine serum albumin is digested by rat yolk sacs exclusively intracellularly, within lysosomes.
A role for bicarbonate in the regulation of mammalian glutamine metabolism
Authors: Baverel G , Lund P
Published: August 2015

1. The concentration of HCO3- (independent of any change of pH) exerts different effects on glutamine metabolism in rat kidney-cortex tubules, hepatocytes and enterocytes.2. In kidney tubules HCO3- (10.5-50 MM) has no effect on glutaminase (EC 3.5.1.2), whereas glutamate dehydrogenase (EC 1.4.1.3) is inhibited as HCO3- concentration is increased. The result is that flux through the entire glutamate-to-glucose pathway is inhibited by increasing HCO3- concentrations. A large proportion (more than 30%) of the glutamine removed undergoes complete oxidation. 3. In hepatocytes, and to a smaller extent in enterocytes, HCO3- is an accelerator of glutaminase. Synthesis of glucose and urea from glutamine in hepatocytes increases as HCO3- concentration is increased. Calculations show that fumarate, formed via aspartate aminotransferase and arginino-succinate lyase, is the precursor of the glucose. There is no complete oxidation of the carbon skeleton of glutamine in hepatocytes. 4. Leucine at near-physiological concentrations (0.1-1 mM) is an accelerator of glutaminase in hepatocytes, but not in kidney tubules or in enterocytes. 5. The results are discussed in relation to regulation of acid/base balance in vivo.
Quantitative analysis of hyaluronate in nanogram amounts
Authors: Tengblad A
Published: August 2015

A radiosorbent technique allowing the specific analysis of 10 ng of hyaluronate has been developed.
Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions
Authors: Nieduszynski I A , Sheehan J K , Phelps C F , Hardingham T E , Muir H
Published: August 2015

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 × 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 × 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol.wt. 670 000), an average Kd of 12 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.
Immuno-biochemical studies of a non-histone chromosomal protein in embryonic and mature chick oviduct
Authors: Teng C T , Teng C S
Published: August 2015

A non-histone chromosomal 95K protein (of mol.wt. 95 000) from hen oviduct was isolated and purified for antibody induction in the rabbit. Immuno-micro-complement-fixation and biochemical techniques were used to probe the presence of 95K protein in the oviduct chromatin of the embryonic and immature chick and the hen. The antiserum against 95K protein did not react with high-mobility-group proteins 1 and 2 obtained from oviduct, brain and liver, nor with histones. After limited digestion of chromatin with nucleases, until 10% DNA was hydrolysed, a small amount of 95K protein was released. Thus the 95K protein is probably not located in the region of chromatin that is sensitive to nuclease digestion. The amount of 95K protein in immature chick oviduct chromatin is less than that in the mature hen oviduct. However, the amount of 95K protein in the immature chick oviduct was increased after oestrogen administration. The presence of 95K protein in embryonic oviduct was detected in the 10-, 12-, 15- and 18- day chick embryo. The quantity of this protein increased with the age of the embryo and reached its highest value in the chromatin of the hen oviduct.
The distribution of iron between the metal-binding sites of transferrin human serum
Authors: Williams J , Moreton K
Published: August 2015

The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250-256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at −15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction.
Partial purification and characterization of a new intracellular β-glucosidase of Trichoderma reesei
Authors: Inglin M , Feinberg B A , Loewenberg J R
Published: August 2015

A new intracellular beta-glucosidase was isolated from Trichoderma reesei. It was sequentially purified by (NH4)2SO4 precipitation and chromatography and rechromatography on Sephadex G-150. The enzyme has a mol.wt. of 98 000, optimal activity at pH 6.5, pI 4.4 and Km values of 6.7 mM and 3.3 mM for sophorose and cellobiose respectively. Possible functions of the enzyme may be regulation of cellulase induction and/or to serve as a proenzyme.
Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside
Authors: Hechtman Peter , Kachra Zarin
Published: August 2015

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of Gm2 ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem.55, 315–324; Hechtman & Leblanc (1977) Biochem. J.167, 693–701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of Gm2 ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-β-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of Gm2 ganglioside decrease the Km for Gm2 ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and Gm2 ganglioside was obtained by comparing gel-filtration profiles of 3H-labelled GM2 ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and GM2 ganglioside is the preferred substrate for enzymic hydrolysis.
Comparison of bone and osteosarcoma adenylate cyclase. Effects of Mg2+, Ca2+, ATP4− and HATP3− in the assay mixture
Authors: Rodan Sevgi B. , Golub Ellis E. , Egan John J. , Rodan Gideon A.
Published: August 2015

The effects of Mg2+ and Ca2+ on bone and osteosarcoma adenylate cyclase were investigated. The concentrations of the cations and other ionic species in the assay mixture were calculated by solving the simultaneous equations describing the relevant ionic interactions (multiple equilibria). We re-examined the effects of HATP3− and ATP4− on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP2−, and their ratio to MgATP2− is constant if Mg2+ and H+ concentrations are unchanged; (ii) Mg2+ addition decreased the ratio of the minor species to MgATP2− and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP3− or ATP4−. On the other hand, kinetic analysis of Mg2+ effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP2−) site, with apparent Km values of approximately 1 and 8mm; (ii) that the low affinity increased towards the higher one when the enzyme activity rose as a result of increased substrate or guanine nucleotide concentrations, this effect being less pronounced in tumour; (iii) conversely, that two apparent affinities for MgATP2− merged into one at high Mg2+ concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg2+ as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca2+ effects showed: (i) competition with Mg2+ at the metal site (Ki 20μm for bone and 40μm for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca2+ binding after substrate binding. These findings support the existence of interacting enzyme complexes, losing co-operativity at increased enzyme activity. They also indicate a potential physiological role for Ca2+ in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.
Reactivation and aging of neurotoxic esterase inhibited by a variety of organophosphorus esters
Authors: Clothier B , Johnson M K
Published: August 2015

1. It was proposed [Johnson (1974) J. Neurochem. 23, 785-789] that both inhibition of neurotoxic esterase of nervous tissue and subsequent ‘aging’ of the inhibited esterase are necessary events in the pathogenesis of organophosphate-induced delayed neuropathy: aging has now been demonstrated with a number of neurotoxic compounds. 2. Reactivation by KF was observed for hen brain neurotoxic esterase inhibited by 14 organophosphates and phosphonates, and time-dependent loss of reactivatibility (aging) occurred in every case. 3. For five other compounds no reactivation occurred and aging could not therefore be established, but independent evidence for two compounds suggests that aging was rapid. 4. Half-lives of aging of neurotoxic esterase inhibited by phosphates ranged from less than 1 min to 10 min, and for phosphonates the range was 3-600 min. 5. The relationship of these findings to the mechanism of toxicity and to the prospects of therapy are considered. 6. Aging occurred rapidly with aryloxy and linear alkoxy groups attached to phosphorus and slowly with a highly branched alkoxy substituent: these effects seem incompatible with an SN1 (dealkylation) mechanism.
The preparation of calmodulins from barley (Hordeum sp.) and basidiomycete fungi
Authors: Grand R J , Nairn A C , Perry S V
Published: August 2015

1. Calmodulin-like proteins were purified from the fruiting bodies of higher (basidiomycete) fungi and barley (Hordeum sp.) shoots. 2. These calmodulins have electrophoretic mobilities on 10% (w/v) polyacrylamide gels at pH 8.3 in the presence of 6 M-urea and at pH 8.3 in the presence of 0.1% sodium dodecyl sulphate similar to that of bovine brain calmodulin. They interacted with rabbit skeletal-muscle troponin I in the presence of Ca2+. 3. Barley and fungal calmodulins activated myosin light-chain kinase and phosphodiesterase in the presence of Ca2+, although the amounts needed were at least an order of magnitude greater than is required to produce the same effect with mammalian calmodulin. 4. Amino acid analyses indicated a number of differences from the mammalian protein, most notably the absence of trimethyl-lysine. 5. By using 125I-labelled calmodulin, a small amount of calmodulin-binding protein was detected in homogenates of barley and fungi. 6. No protein corresponding to calmodulin could be found in Escherichia coli or yeast, although a relatively high concentration of a protein that bound calmodulin was detected in E. coli by this technique.
Rapid type 2 molybdenum(V) electron-paramagnetic resonance signals from xanthine oxidase and the structure of the active centre of the enzyme
Authors: Malthouse J P , Gutteridge S , Bray R C
Published: August 2015

Rapid type 2 molybdenum(V) e.p.r. signals from reduced functional xanthine oxidase have been further investigated. These signals, which show strong coupling of two protons to molybdenum, have been obtained under a variety of new conditions: specifically either at pH 8.2 in the presence of borate ions, or at pH 10.1-10.7 with or without various other additions. Parameters of the signals were obtained with the help of computer simulations. In at least some of these signals, the coupled protons must be located on the enzyme rather than on bound species. The relationship between type 1 and type 2 Rapid signals is discussed. They may represent geometrical isomers, or alternatively, hydroxyl uptake as a ligand of molybdenum may be involved in formation of type 2 species.
A comparative study of the early effects of phenobarbital and 3-methylcholanthrene on the synthesis and transport of ribonucleic acid in rat liver
Authors: Kumar A , Rao M R S , Padmanaban G
Published: August 2015

At 2-3 h after phenobaribtal administration, the drug has no effect on nucleoplasmic RNA synthesis and decreases nucleolar RNA synthesis. However, at this time there is an increase in the labelling of cytoplasmic poly(A)-containing RNA, even though there is decreased labelling of total polyribosomal RNA. The decrease in labelling of nucleolar and total polyribosomal RNA owing to phenobarbital is a transient phenomenon. Under similar conditions, 3-methylcholanthrene has no effect on nucleolar RNA synthesis, but leads to an increase in synthesis of nucleoplasmic and cytoplasmic poly(A)-containing RNA. Cytosol isolated from phenobarbital-treated, but not from 3-methyl-cholanthrene-treated, animals facilitates an enhanced transport of RNA from nuclei. At the time points investigated, 3-methylcholanthrene or its metabolite shows a 10-15-fold higher concentration in the chromatin than that of phenobarbital or its metabolite. It is suggested that the primary effect of phenobarbital is at the cytoplasmic level, promoting the transport of RNA from the nuclei, which can act as a trigger for enhanced transcription at later periods. 3-Methylcholanthrene or its metabolite directly binds to the chromatin and evokes a selective transcriptional response.
Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver
Authors: MacNicholl A D , Wusteman F S , Winterburn P J , Powell G M , Curtis C G
Published: August 2015

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.
Hormone-initiated maturation of rat liver mitochondria after birth
Authors: Sutton R , Pollak J K
Published: August 2015

1. The injection of adrenaline, glucagon or cyclic AMP into foetal rats in utero initiates the maturation of energy transduction in rat liver mitochondria before birth. 2. The injection of the beta-blocker, propranolol, prevents this maturation process. 3. The maturation of mitochondrial energy transduction is measured in terms of the increase in the respiratory control index and mitochondrial adenine nucleotide concentration. 4. It is postulated that the actions of the hormones, acting through cyclic AMP, affect glycogenolysis and glycolysis to give rise to transient localized high concentrations of ATP. 5. It is the ATP that acts as the molecular trigger, effecting mitochondrial maturation.
Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase
Authors: Shigeoka S , Nakano Y , Kitaoka S
Published: August 2015

Euglena gracilis was found to contain a peroxidase that specifically require L-ascorbic acid as the natural electron donor in the cytosol. The presence of an oxidation-reduction system metabolizing L-ascorbic acid was demonstrated in Euglena cells. Oxidation of L-ascorbic acid by the peroxidase, and the absence of ascorbic acid oxidase activity, suggests that the system functions to remove H2O2 in E. gracilis, which lacks catalase.
The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase
Authors: Lemon M , Methven P , Bhoola K
Published: August 2015

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.
Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes
Authors: Francis G L , Ballard F J
Published: August 2015

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte “ghosts” showed slight activity and erythrocyte and reticulocyte “ghosts” were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.
Interactions between vasopressin and glucagon on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats
Authors: Williamson D H , Ilic V , Tordoff A F , Ellington E V
Published: August 2015

Vasopressin (10nM) inhibited ketogenesis (56%) in hepatocytes from fed rats when oleate (1 mM) was the substrate, but had no effect with butyrate (10mM). The hormone increased the accumulation of lactate and stimulated the esterification of [1(-14)C]oleate (70%). These effects of vasopressin were reversed by glucagon (10 nM). The physiological implications of these findings are discussed.
Tryptophan pyrrolase in haem regulation. The relationship between the depletion of rat liver tryptophan pyrrolase haem and the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide
Authors: Badawy A A B , Morgan C J
Published: August 2015

Rat liver tryptophan pyrrolase haem is maximally depleted at 30 min after administration of a 400 mg/kg dose of 2-allyl-2-isopropylacetamide. This depletion lasts for 24 h, by which time 5-aminoleevulinate synthase activity becomes maximally enhanced. 2. though the above maximum depletion of pyrrolase haem (at 0.5h) is also produced by a 100 mg/kg dose of the porphyrogen, this does not enhance synthase activity at 24 h. It and smaller doses, however, cause a smaller but earlier enhancement of synthase activity (maximum at 2 h) and produce a similarly short-lived deplation of pyrrolase haem. 3. The depletion of pyrrolase haem and the enhancement of synthase activity by the porphyrogen are inhibited by compound SKF 525-A and phenazine methosulphate, and are potentiated by nicotinamide but not by phenobarbitone. Phenazine methosulphate and nicotinamide also exert opposite effects on hexobarbital sleeping-time. 4. 2-Allyl-2-isopropylacetamde also the depletes pyrrolase haem in vitro. It does so in liver homogenates of control rats in the presence, and in those of phenobarbitone-treated rats in the absence of added NADPH. 5. A discussion of the present results in relation to previous work with other haemoproteins suggests that, whereas cytochrome P-450 (haem) is primarily involved in the production of the active (porphyrogenic) metabolite(s) of 2-allyl-2-isopropylacetamide, the haem pool used by tryptophan pyrrolase may play an important role in the effects of this compound on haem biosynthesis.
Perinatal developmental changes in hepatic UDP-glucuronyltransferase
Authors: Goldstein R B , Vessey D A , Zakim D , Mock N , Thaler M
Published: August 2015

Postnatal developmental changes in hapatic microsomal UDP-glucuronyltransferase were studied in the rat. The previously reported postnatal decline in the capacity of microsomal fractions to glucuronidate p-nitrophenol was found to be observable in unperturbed preparations only at non-saturating concentrations of the substrate UDP-glucuronic acid. At saturating concentrations of UDP-glucuronic acid, activity is identical in newborns and adults. Kinetic analysis revealed that the enzyme from liver of newborns has a much higher affinity for UDP-glucuronic acid than does the enzyme in adults, but the same activity at Vmax. On the other hand, the enzyme from adult liver microsomal fractions can be activated by the physiological allosteric effector UDP-N-acetylglucosamine, whereas the enzyme from newborns is largely unaffected by it. Thus it appears that the number of enzyme active sites is not changing; rather, the enzyme is maturing to a more highly regulable form. There were also differences between the enzymes in newborns and adults in their response to perturbation of the membrane-lipid environment by detergent and phospholipase A. Possible interpretations of these differences are discussed.
The detection of messenger ribonucleic acid sequences in heterogeneous nuclear ribonucleic acid fractions of the oestrogen-stimulated rat uterus
Authors: Aziz S , Knowler J T
Published: August 2015

cDNA (complementary DNA) complementary to the abundant sequences of mRNA isolated from oestrogen-stimulated uterus was hybridized to polyadenylated and non-polyadenylated uterine hnRNA (heterogeneous nuclear RNA). High-molecular-weight polyadenylated hnRNA, isolated under denaturing conditions, was able to saturate the cDNA, but complementary sequences were low abundance when compared with homologous mRNA. The mRNA sequence content of the polyadenylated hnRNA increased considerably during oestrogen-induced growth of the uterus. Non-polyadenylated hnRNA also contained sequences complementary to the cDNA.
Regulation of nitrogenase A and R concentrations in Rhodopseudomonas capsulata by glutamine synthetase
Authors: Yoch D C
Published: August 2015

Nitrogen-starved purple non-sulphur bacteria have an active unregulated form of nitrogenase (nitrogenase A); however, the nitrogenase of a glutamine synthetase-negative mutant of Rhodopseudomonas capsulata, when nitrogen-starved, was predominantly inactive and required activation by Mn2+ and activating-factor protein. This regulatory form of nitrogenase has been called nitrogenase R. Treatment of wild-type cells (containing nitrogenase A) with methionine sulphoximine, an inhibitor of glutamine synthetase, converted the enzyme into nitrogenase R. Glutamine synthetase thus appears to control the intracellular concentrations of nitrogenase A and R and in this way regulates nitrogenase activity in the photosynthetic bacterium.
Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli
Authors: Ambrose-Griffin Mary C. , Danson Michael J. , Griffin William G. , Hale Geoffrey , Perham Richard N.
Published: August 2015

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.
Studies of inhibition of rat spermidine synthase and spermine synthase
Authors: Hibasami Hiroshige , Borchardt Ronald T. , Chen Shiang Yuan , Coward James K. , Pegg Anthony E.
Published: August 2015

1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5′-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70–98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5′-Methylthioadenosine, 5′-ethylthioadenosine and 5′-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10–15μm and 50% inhibition of spermidine synthase at 30–45μm. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20μm respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5′-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.
Sheep gastric mucins as a source of blood-group-I and -i antigens
Authors: Wood E , Hounsell E F , Langhorne J , Feizi T
Published: August 2015

Gastric-mucosal glycoproteins of sheep have been shown to express the blood-group-I and -i antigens. The highest blood-group-I activities were found in glycoproteins lacking in blood-group-A and -H activities. In antigenically very active glycoprotein preparations, approx. 25% of the macromolecules expressed various blood-group-I and -i antigens, and these could be enriched by affinity chromatography with an anti-(blood group I) immunoadsorbent column. Additional blood-group-I and -i activities could be revealed by one cycle of Smith degradation of blood-group-A-active sheep gastric glycoproteins. Thus sheep gastric mucins are an abundant source of oligosaccharides with blood-group-I- and -i-active sequences.
Non-classical inhibition of uricase by cyanide
Authors: Conley T G , Priest D G
Published: August 2015

The interactions of Aspergillus flavus uricase with the substrates O2 and urate and the inhibitors xanthine, cyanide, periodate and hydroxylamine were investigated. Under equilibrium conditions O2 does not bind directly to the enzyme, and the absence of O2 had no effect on either the binding stoicheiometry or binding constant for xanthine, as measured by equilibrium dialysis and microcalorimetry. Cyanide, periodate and hydroxylamine inhibit uricase in a non-classical manner. A decrease in initial velocity to a steady-state inhibited velocity can be observed on a time scale of minutes. The time-dependence, which is unaltered by prior incubation with the inhibitors, is consistent with a first-order transition. Rate constants for induction of inhibition are linearly dependent on inhibitor concentration, but independent of urate and O2 concentrations. Radioactively labelled urate forms a stable but reversible complex with uricase in the presence of cyanide and O2. These results were used to deduce the nature of enzyme-bound intermediates and thus for the proposal of a novel mechanism for cyanide inhibition.
Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, β-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase
Authors: Bardsley W G , Leff P , Kavanagh J , Waight R D
Published: August 2015

The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS)
Preparation and characterization of chemically defined oligomers of rabbit immunoglobulin G molecules for the complement binding studies
Authors: Wright J K , Tschopp J , Jaton J C
Published: August 2015

Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at −70 degrees C.
Formulation of N-acetylputrescine and N1-acetylspermidine in cultured human lymphocytes
Authors: Menashe M , Faber J , Bachrach U
Published: August 2015

N-Acetylputrescine and N1-acetylspermidine were synthesized from putrescine in cultured human lymphocytes. They were identified by paper chromatography and t.l.c. Acetylputrescine was also identified by mass spectrometry. N-Acetylputrescine and N1-acetylspermidine were formed in untreated cells and in lectin-transformed cells.
Metabolism of hepatic haem and ‘green pigments’ in rats given 2-allyl-2-isopropylacetamide and ferric citrate. A new model for hepatic haem turnover
Authors: Bonkowsky Herbert L. , Healey John F. , Sinclair Peter R. , Mayer Yash P. , Erny Raymond
Published: August 2015

We have studied effects of single doses of 2-allyl-2-isopropylacetamide and ferric citrate on hepatic haem turnover in rats. Haem was pre-labelled by intraperitoneal administration of 5-amino-[4-14C]laevulinate 4h before other treatments. Computer-assisted analysis of the haem decay curve showed that at least two exponential components were involved implying two haem pools. In control rats the size of the rapidly-turning-over pool was 38% of the total. Treatment with 2-allyl-2-isopropylacetamide alone resulted in a 2.3-fold increase in the fractional size of this pool. Treatment with ferric citrate alone increased the size of this pool 1.8-fold; treatment with both agents together had no measurable effect beyond that produced by 2-allyl-2-isopropylacetamide. The apparent rate constant for disappearance of labelled haem from the first pool was not affected by treatment with 2-allyl-2-isopropylacetamide or ferric citrate, indicating that these treatments affect hepatic haem turnover primarily by altering the distribution of haem synthesized in the liver. The increased haem degradation after treatment with 2-allyl-2-isopropylacetamide alone was associated with the accumulation of ‘green pigments’ in the liver. These pigments were detectable 1.5h after drug treatment; their total amount increased steadily for 28h and then declined. Despite this increase in amount, radioactivity of the ‘green pigment’ fraction fell rapidly in a biphasic fashion. Some of the radioactivity that initially was found in the ‘green pigment’ fraction was later found in an aqueous fraction, not extractable by acidic ethyl acetate. Rats given ferric citrate together with 2-allyl-2-isopropylacetamide accumulated less ‘green pigments’.
Studies on sex-organ development. Effect of hormones on ornithine decarboxylase activity in the developing chick ovary
Authors: Teng Ching Sung , Teng Christina T.
Published: August 2015

The response of ornithine decarboxylase activity to hormones in the embryonic left ovary was measured throughout the stages of development. During the early stage of ovarian development (9th day of incubation), the ornithine decarboxylase activity (in terms of pmol CO2/30min per mg of protein) was high (766); it decreased from the 10th to the 12th day (575–239), increased slightly from the 13th to the 15th day (306) and finally fell to a low value (192–20) from the 18th day of development to birth. Administration of an optimal dose of oestrogen to the 9–10-day embryo stimulated the ovarian ornithine decarboxylase activity by 48–53%. If the same dose of oestrogen was administered to the 15–18-day embryo, the ovarian enzyme activity was suppressed by 32–43%. This biphasic response to oestrogen for enzyme induction is characteristic of the developing ovary and is not observed in other genital organs of the chick. In the early developmental stage (9–10th day) testosterone has no effect on ovarian ornithine decarboxylase activity, but in the late stage testosterone inhibits the activity by 41%. Organ culture techniques have been used to test the ovarian response to lutropin (luteinizing hormone). Lutropin stimulated ornithine decarboxylase activity by approx. 99–155% in the ovary of the early embryonic stage (10–13th day), and by 175–200% in the ovary of the late embryonic stage (15–18th day). The alteration in enzyme activity in the ovary as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. The relationship of ornithine decarboxylase activity to the morphological and biochemical changes in the developing ovary is discussed.
Ribonucleotide reductase activity in vitamin B12-deficient Euglena gracilis
Authors: Carell E F , Seeger J W
Published: August 2015

The ribonucleotide reductase activities in vitamin B12-sufficient and -deficient cells of Euglena gracilis were measured. We found that the cells progress into vitamin B12 deficiency the enzyme activity increases, reaching a maximum value of 20-fold in advanced deficiency. No signigicant differences in the activities were found to result as a consequence of different growth conditions. We propose that the increased activity in vitamin B12-deficient cells is due to an increase in enzyme protein.