bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat α-foetoprotein. Purification, physicochemical characterization, oestrogen-binding properties and chemical modification of the thiol group
Authors: Versée Vera , Barel André O.
Published: August 2015

1. Rat α-foetoprotein, an oestrogen-binding foetal globulin, was isolated in large quantities from amniotic fluid and serum by preparative electrophoresis on polyacrylamide slab gels or by chromatography on an immunoadsorbent column. Subsequently the two electrophoretic forms of this protein were separated by electrophoresis on the same medium. 2. Both forms were found to show identical binding with oestradiol. From the extrinsic fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonic acid it was shown that the polarity of the binding site is practically identical for both forms. One residue of tryptophan was determined for both forms. The two electrophoretic variants display the same amount of secondary structure as demonstrated by circular dichroism. 3. The affinity of total α-foetoprotein for oestradiol as a function of pH was studied by using a Sephadex G-25 gel-equilibration method. Maximal binding occurred at pH8.5. Only a fractional number of binding sites per molecule could be measured at pH7.4, whereas at higher pH the number of sites was very close to unity. There was no significant effect of pH on the value of the association constant (Ka=4.3×107±1.2×107m−1). 4. Displacement experiments of bound labelled oestradiol with various steroids have permitted investigation of the specificity of α-foetoprotein. This foetal globulin binds rather strongly compounds that display the rigid structure of the oestratriene skeleton (oestradiol, oestrone). Diminished binding for diethylstilboestrol and a diethylstilboestrol affinity label was observed. No binding was measured with a more flexible structure such as hexoestrol [4,4′-(1,2-diethylethane-1,2-diyl)bisphenol]. 5. Chemical modification of cysteine residues of α-foetoprotein with two alkylating reagents [iodoacetic acid and 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid] has very little effect on the oestrogen binding. It is suggested that the oestrogen-binding site does not contain a cysteine residue. From the kinetics of alkylation and from the fluorescence properties of the chemically bound thiol reagent 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid], it was demonstrated that the very-slow-reacting thiol group is probably located in a non-polar region of the molecule.
Purification and some properties of tartrate-sensitive acid phosphatase from rabbit kidney cortex
Authors: Helwig J J , Farooqui A A , Bollack C , Mandel P
Published: August 2015

Two forms of tartrate-sensitive acid phosphatases (EC 3.1.3.2) were purified from rabbit kidney cortex by a multiple-column-chromatography method. The basic form constituted 90% of the enzyme and migrated as a single band of protein on polyacrylamide-gel electrophoresis. The proteins contaminating the acidic form did not exceed 5% of the total protein. The specific activity towards p-nitrophenyl phosphate was 12 mumol/min per mg for the basic form and 0.7 mumol/min per mg for the acidic form. The basic form of the enzyme differs from the acidic form in its heat-stability, Km values, inhibition rates by tartrate and fluoride and substrate specificities. Relative to p-nitrophenyl phosphate hydrolysis rate, the acidic form hydrolysed a variety of physiological monophosphate esters, whereas the basic form hydrolysed only CMP and phosphoenolpyruvate. Bacterial neuraminidases had no effect on the activity and mobility of the acidic form on polyacrylamide-gel electrophoresis. Both forms have the same molecular weight (101000 +/- 4000) and are probably composed of two identical subunits. The question whether the two forms of the enzyme are different proteins or whether one is a modified form of the other is discussed.
Incorporation of oxygen-18 into the 25-position of cholecalciferol by hepatic cholecalciferol 25-hydroxylase
Authors: Madhok T C , Schnoes H K , DeLuca H F
Published: August 2015

The oxygen enzymically inserted as a hydroxy function by rat liver post-mitochondrial fraction into the 25-position of cholecalciferol to giver 25-hydroxycholecaliferol is derived exclusively from molecular O2. Therefore like the other two cholecalciferol hydroxylases, i.e. 25-hydroxycholecalciferol 1alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, the cholecalciferol 25-hydroxylase is also a mono-oxygenase (‘mixed-function oxidase’).
Analysis of glycosaminoglycans in urine by using acridine orange fluorescence
Authors: Diakun G P , Edwards H E , Allen J C , Phillips G O
Published: August 2015

The fluorescence technique described here utilizes the electrostatic interaction between the polyanionic sites of glycosaminoglycans and the cationic dye Acridine Orange to analyse urinary glycosaminoglycans from patients suffering from mucopolysaccharidoses. The basis of the titration is the decrease in the fluorescence of free Acridine Orange that occurs when it is bound to polyanions. The effect of the presence of possible interfering materials such as salt, proteins and trace materials in urine was evaluated. This fluorescence technique is technically simple.
The kinetics and thermodynamics of the reaction of solid-state fully reduced membrane-bound cytochrome oxidase with carbon monoxide as studied by dual-wavelength multichannel spectroscopy and flash photolysis
Authors: Clore G M , Chance E M
Published: August 2015

1. The results of non-linear optimization studies on the mechanism of reaction of solid-state fully reduced membrane-bound cytochrome oxidase with CO over the 178–203 K range are presented. The analysis is carried out on data obtained by dual-wavelength multichannel spectroscopy at three wavelength pairs (444–463 nm, 590–630 nm and 608–630 nm), which yield three distinct progress curves. The only model that satisfies the triple requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters is a two-species sequential mechanism: flash photolysis yields unliganded cytochrome oxidase and free CO, which then recombine to form species Ic; Ic is then converted into species IIc, which is identical with the cytochrome oxidase-CO complex existing before flash photolysis. All the thermodynamic parameters describing this model are calculated. 2. On the basis of the data obtained from this paper, together with data from potentiometric studies, magnetic susceptibility measurements and i.r. spectroscopy, the chemical identity of the species is suggested.
p-NN′-phenylenebismaleimide, a specific cross-linking agent for F-actin
Authors: Knight P , Offer G
Published: August 2015

Covalent cross-links can be inserted between the subunits of F-actin by using p-NN′-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN′-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.
A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation
Authors: Palmer D N , Rabin B R , Williams D J
Published: August 2015

The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.
Synthesis of 1 α-hydroxy[7-3H]cholecalciferol and its metabolism in the chick
Authors: Edelstein S , Noff D , Freeman D , Sheves M , Mazur Y
Published: August 2015

1. 1 alpha-Hydroxy[7-3H]cholecalciferol (specific radioactivity of 2-Ci/mmol) was synthesized, and its metabolism in chicks studied. 2. 1 alpha-Hydroxy[7-3H]cholecalciferol was metabolized very rapidly in the chick to 1 alpha,25-dihydroxy[7-3H]cholecalciferol and to a metabolite less polar than 1 alpha-hydroxycholecalciferol. Intestine exhibited highest accumulation of 1 alpha-25-dihydroxy[7-3H]cholecalciferol, and liver exhibited highest accumulation of the non-polar metabolite. 3. Tissue uptake of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolites in chicks that were dosed continuously for 16 days with 1 alpha-hydroxy[7-3H]cholecalciferol did not exceed by very much that observed in tissues obtained from chicks that were dosed with a single injection of 1 alpha-hydroxy[7-3H]cholecalciferol 24 h before killing, except for liver and kidney. 4. Lowest accumulation of metabolites was noted in muscle and bone, and for the latter, highest uptake of 1 alpha,25-dihydroxy[7-3H]cholecalciferol was noted in the epiphysial periosteum and the metaphysis. 5. Formation of 1 alpha,24,25-trihydroxy[7-3H]cholecalciferol was not observed in the chicks that were dosed continuously with 1 alpha-hydroxy[7-3H]cholecalciferol, despite the fact that plasma calcium and phosphorus were normal and despite the presence of renal 24-hydroxylase activity. 6. The vitamin D status of the chicks did not appear to affect the metabolic profile of the administered 1 alpha-hydroxy[7-3H]cholecalciferol.
Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid
Authors: Hughes Julia , Mellows Graham
Published: August 2015

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RCrel, whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNAIle. Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.
Evidence for a role of insulin in the regulation of lipogenesis in lactating rat mammary gland. Measurements of lipogenesis in vivo and plasma hormone concentrations in response to starvation and refeeding
Authors: Robinson A M , Girard J R , Williamson D H
Published: August 2015

Fatty acid synthesis in the mammary gland of lactating rats in vivo was 5-fold higher than in the liver. Starvation decreased fatty acid synthesis in the gland 50-fold, whereas refeeding for 2h completely reversed this change. The plasma insulin concentration decreased 2-fold in starvation and was restored to the fed-rat value on refeeding. Glucagon and prolactin concentrations did not always change in parallel with lipogenesis, suggesting that insulin may be a regulator of this process in the gland.
An analysis of errors in estimation of the rate of protein synthesis by constant infusion of a labelled amino acid
Authors: Garlick P J
Published: August 2015

Rates of protein synthesis in tissues can be calculated from the specific radioactivity of free and protein-bound amino acids at the end of a constant infusion of a labelled amino acid (Garlick, Millward & James (1973) Biochem. J. 136, 935–945]. The simplifying assumptions used in these calculations have been criticized [Madsen, Everett, Sparrow & Fowkes (1977) FEBS Lett. 79, 313–316]. A more detailed analysis using a programmable desk-top calculator is described, which shows that the errors introduced by the simplifying assumptions are small, particularly when the specific radioactivity of the free amino acid rises rapidly to a constant value.
Ovalbumin digestion by human pepsins 1, 3 and 5
Authors: Walker V , Taylor W H
Published: August 2015

1. Of the three major human pepsins, pepsin 1 has greater proteolytic activity towards ovalbumin than has pepsin 3. Pepsin 5 has low activity towards this substrate. 2. Proteolytic pH-activity curves show only on pH maximum, about pH 1.4 for pepsin 1, pH 1.4–1.5 for pepsin 3 and pH 1.2–1.4 for pepsin 5. The curve for pepsin 3 has a shoulder between pH 2.4 and 3.4. 3. The rate of digestion of ovalbumin by pepsin 1 is approximately three times slower than are those of bovine haemoglobin or human globin. 4. The results suggest that there may be a physiological advantage in having more than one pepsin.
The effects of food deprivation on protein turnover and nucleic acid concentrations of active and immobilized extensor digitorum longus muscles of the rat
Authors: Goldspink D F
Published: August 2015

Deprivation of food caused significant changes in the weight, protein content, protein turnover and RNA concentrations of the extensor digitorum longus muscle. Simultaneous immobilization to render the muscle inactive did not make the tissue any more susceptible to the effects of starvation. In contrast, immobilization in a stretched state resulted in less muscle wasting after deprivation of food.
Activation of pyruvate dehydrogenase in the perfused rat liver by vasopressin
Authors: Hems D A , McCormack J G , Denton R M
Published: August 2015

The proportion of pyruvate dehydrogenase in its active form is doubled in rat liver within 5 min of addition of vasopressin to the perfusing medium.
The effects of cortisol, corticotropin and thyroxine on the synthesis of glycerolipids and on the phosphatidate phosphohydrolase activity in rat liver
Authors: Glenny Helen P. , Brindley David N.
Published: August 2015

1. Male rats were injected daily for 5 days with 0.15m-NaCl, corticotropin, cortisol or l-thyroxine and the rates of glycerolipid synthesis were measured in the livers after intraportal injection of [14C]palmitate and [3H]glycerol. 2. Injection of all three hormones decreased the rates of body-weight gain. 3. Cortisol treatment increased the weight of the liver relative to body weight. 4. Thyroxine treatment increased the relative rate of triacylglycerol synthesis from [3H]glycerol and decreased the relative accumulation of 3H and 14C in diacylglycerol. It did not significantly alter the accumulation of these isotopes in phosphatidate nor the activity of the soluble phosphatidate phosphohydrolase in the total liver. However, this activity increased by 1.5-fold when expressed relative to the soluble protein of the liver. The increased triacylglycerol synthesis appears to be related to a general increase in the turnover of fatty acids in the liver. 5. Treatment with cortisol and corticotropin increased the relative rate of triacylglycerol synthesis from [3H]glycerol, decreased the accumulation of 3H in phosphatidate and increased the flux of both isotopes from phosphatidate to diacylglycerol. This appeared to be caused by the increased activity of the soluble phosphatidate phosphohydrolase that was observed in the livers of the cortisol-treated rats. 6. It is proposed that cortisol could be directly or indirectly involved in increasing the activity of hepatic phosphatidate phosphohydrolase in starvation, diabetes, laparotomy, subtotal hepatectomy, liver damage, ethanol feeding and in obesity. This enzyme adaptation could contribute to the potential of the liver to increase its synthesis and accumulation of triacylglycerols or to secrete very-low-density lipoproteins.
Relative rates of turnover of subunits of mitochondrial proteins
Authors: Walker J H , Burgess R J , Mayer R J
Published: August 2015

1. The double-isotope concept [Arias, Doyle & Schimke (1969) J. Biol. Chem. 244, 3303–3315] for the measurement of protein turnover was used to estimate the turnover rates of protein subunits from rat liver submitochondrial fractions resolved by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. NaH14CO3 and [5-3H]arginine were used as first and second precursors respectively. 2. Marked heterogeneity of protein subunit turnover rates is seen for protein subunits from water-soluble, salt-soluble and Tween 20-soluble mitochondrial proteins. 3. Much lower heterogeneity is seen in the turnover of protein subunits in Triton X-100-soluble material not binding to DEAE-cellulose at low ionic strength. The relative rates of turnover of proteins in this fraction are lower than for proteins in any other submitochondrial fraction. This fraction contains the integral membrane proteins. 4. Incorporation of [3H]arginine into subunits of the cytochrome oxidase complex is greatest for subunits with molecular weights in excess of 20000. 5. No correlation is seen between protein subunit size and the rate of turnover of the protein subunits in any of the submitochondrial fractions.
The influence of respiratory state on monoamine oxidase activity in rat liver mitochondria
Authors: Smith G S , Reid R A
Published: August 2015

Changes in the respiratory state of rat liver mitochondria caused significant changes (up to 10-fold) in the rates of oxidative deamination of tyramine, indicating interactions between the inner coupling membrane and the monoamine oxidase sites in the outer membrane, and suggesting the possibility that monoamine oxidase is regulated by the thermodynamic state of the mitochondria.
Characterization of a proteolytic-enzyme inhibitor with allergenic activity. Multiple functions of a parasite-derived protein
Authors: Willadsen P , Riding G A
Published: August 2015

1. A trypsin inhibitor from the tick Boophilus microplus was purified by ion-exchange chromatography and gel filtration. 2. It is pure by the criteria of constant specific activity on gel filtration and by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate. 3. The protein undergoes reversible polymerization, dissociating at low pH. 4. The apparent molecular weight measured by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate is 18,500. 5. Inhibition of trypsin occurs by formation of a 1 :1 molar complex. 6. Chymotrypsin is also inhibited, though the dissociation constant of the complex formed is larger than with trypsin. The protein possesses independent sites for the inhibition of chymotrypsin and trypsin. 7. The inhibitor preparation gives an immediate hypersensitivity reaction on intradermal injection into cattle that have been exposed to the tick. The allergenic activity is due to the inhibitor protein itself and not to contaminating material, since the two activities were not separated during purification or in two subsequent affinity-chromatography procedures. 8. The hypersensitivity reaction is a true immunological response, since it is found in almost all cattle that have been exposed to the tick, but not in unexposed animals. In addition, passive cutaneous anaphylaxis can be demonstrated with serum from exposed, but not from unexposed, animals.
Removal of cadmium(II) from crystallized ferritin
Authors: Hegenauer J , Saltman P , Hatlen L
Published: August 2015

The cadmium content of crystallized horse spleen ferritin, usually about 2% by weight without special treatment, can be substantially decreased by prolonged dialysis against certain chelating agents, chaotropic ions, or weakly reducing anions. For example, neutral bisulphite buffer (2M) removed 95% of the bound cadmium of crystallization without affecting the iron content, and may thus be valuable for preparing “metal-free” holoferritin for physical-chemical studies.
Compound C2, a product of the reaction of oxygen and the mixed-valence state of cytochrome oxidase. Optical evidence for a type-I copper
Authors: Chance B , Saronio C , Leigh J S
Published: August 2015

Compound C2 is a product of the reaction of O2 and the mixed-valence state of cytochrome oxidase. The mixed-valence state of membrane-bound cytochrome oxidase is obtained at -24 degrees C, by using either ferricyanide or yeast peroxidase complex ES as oxidants, and the configurations of oxidized haem a and its associated copper (a3+Cua2+) and of reduced haem a3 and its associated copper (ac3+.CO.Cua3+) are obtained. The mixed-valence-state cytochrome oxidase mixed with O2 at -24 degrees C and flash-photolysed at -60 to -100 degrees C reacts with O2 and initially forms an oxy compound (A2) similar to that formed from the fully reduced state (A1). Thereafter the course of the reaction differs from that obtained in the fully reduced state, and absorbance increases are observed at 740–750 nm and 609 nm and a decrease at 444 nm, with no increase in absorbance at 655 nm. One possible attribution of the absorbance increases is to charge-transfer interaction between the iron of haem a3 and the copper associated with haem a3, Cua3(2+), having properties of a type-I ‘blue’ copper. A possible attribution of the decrease in absorbance at 444 nm is to liganding of a3(2+). A related explanation is that the 609 nm absorbance involves a charge-transfer interaction of both iron and copper as a mixed-valence binuclear complex, Cua3, having properties of a non-blue copper. Intermediates in addition to Compound C2 are not yet identifiable by chemical or spectroscopic tests. The kinetic and equilibrium properties of Compound C2 are described.
The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase
Authors: Wilton D C
Published: August 2015

1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 × 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195–1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.
N-terminal sequences of uteroglobin and its precursor
Authors: Atger M , Mercier J C , Haze G , Fridlansky F , Milgrom E
Published: August 2015

Translation of uteroglobin mRNA in wheat-germ extract has yielded a precursor protein (pre-uteroglobin) containing an N-terminal extension of 21 amino acid residues. The sequence of this extension and that of the 50 N-terminal amino acid residues of uteroglobin have been determined.
Prolyl 3-hydroxylase and 4-hydroxylase activities in certain rat and chick-embryo tissues and age-related changes in their activities in the rat
Authors: Tryggvason K , Majamaa K , Kivirikko K I
Published: August 2015

Prolyl 3-hydroxylase activity, expressed per unit of extract protein, was much higher in rat kidney cortex than in the lung, liver or skin. A marked decrease in activity was found in the kidney cortex, liver and skin beyond 10 days of age. The ratio of prolyl 3-hydroxylase to 4-hydroxylase activity in the kidney cortex was 13–17 times that in the skin, that in the liver 6–8 times, and that in the lung about twice the value for the skin, there being no changes in this ratio with age. In 16-day chick embryos the highest ratios of prolyl 3-hydroxylase to 4-hydroxylase activity were found in the liver, heart, lens, aorta and kidney, and the lowest ratios in tendon, cartilage, cartilaginous and membranous bone and skin. The results suggest that the differences in the extent of prolyl 3-hydroxylation between various collagens can in part be explained by differences in the amount of prolyl e-hydroxylase activity among different cells.
Ca2+-stimulated ribonuclease. A new marker enzyme of differentiated rat mammary tissues
Authors: Liu D K , Kulick D , Williams G H
Published: August 2015

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.
L-proline transport by newborn rat kidney brush-border membrane vesicles
Authors: Goldmann D R , Roth K S , Langfitt T W , Segal S
Published: August 2015

The transport of L-proline was studied in brush-border membrane vesicles isolated from the kidneys of newborn rats. In contrast with the rapid initial uptake with an ‘overshoot’ observed in adult vesicles, uptake by the newborn vesicle was slow, showed no ‘overshoot’, and proline continued to accumulate at a time when the adult vesicle had already equilibrated. L-Proline transport in the newborn rat occurs by Na+-dependent and independent mechanisms. There appeared to be essentially no uptake by anti-luminal vesicles isolated from newborn rat kidney. These observations may help to explain the prolinuria that occurs in the newborn animal.
Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride
Authors: Hino Y , Asagami H , Minakami S
Published: August 2015

1. The microsomal haem oxygenase activity induced by the administration of CoCl2 was found mainly in the smooth-surfaced microsomal fraction, whereas that of the untreated control animals was widely distributed in smooth-surfaced microsomal, rough-surfaced microsomal and Golgi fractions. 2. When microsomal preparation was incubated and the time course of the distribution of biliverdin between the membranes and the medium was followed, most of the biliverdin formed was found first in the medium. This suggests that the active site of haem oxygenase is exposed on the cytoplasmic surface of the membranes. The possible localization of the enzyme at the outer surface of the membranes was also supported by a digestion experiment with trypsin. The haem oxygenase activity was greatly decreased even at low concentration of the proteinase, which did not affected the NADPH-cytochrome c reductase activity. 3. When microsomal preparation was further fractionated by isopycnic centrifugation in the presence of deoxycholate or by partitioning of sonicated microsomal preparation in aqueous-polymer two-phase systems, most of the haem oxygenase activity was found in a fraction different from the main fraction of the NADH- and NADPH-cytochrome c reductase and NADH–ferricyanide reductase activities. This indicates the different distribution of haem oxygenase from the other enzymes mentioned, on the lateral plane of microsomal membranes, and suggests the different localization of the haem oxygenase system from the electron-transport system linked with cytochrome b5 and cytochrome P-450.
The permeability of rat liver lysosomes to sugars. Evidence for carrier-mediated facilitated diffusion
Authors: Docherty K , Brenchley G V , Hales C N
Published: August 2015

1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.
Interaction between insulin-storage granules and F-actin in vitro
Authors: Howell S L , Tyhurst M
Published: August 2015

Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1–0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.
The effect of starvation on the rate of protein synthesis in rat liver and small intestine
Authors: McNurlan M A , Tomkins A M , Garlick P J
Published: August 2015

1. A method is described that allows for measurement of protein synthesis in liver and intestine in the rat. By injecting a massive amount of [14C]leucine (100 mumol/100 g body wt.) an attempt has been made to over come problems of precursor specific radioactivity and problems arising from the breakdown of labelled protein that are encountered when tracer amounts of amino acids are used. 2. Starvation for 2 days resulted in decline in the rate of total liver protein synthesis from 87%/day to 62%/day. 3. In jejunal mucosa the rate of protein synthesis was 136%/day. This declined to 105%/day after 2 days of starvation.
Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets
Authors: Haslam Richard J. , Lynham James A. , Fox Joan E. B.
Published: August 2015

Human platelets that had been preincubated with 5-hydroxy[3H]tryptamine and [32P]Pi were stirred with various agents; the secretion of 5-hydroxy[3H]tryptamine from platelet granules and the radioactivity of platelet [32P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[3H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E1, which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[3H]tryptamine. Prostaglandin E1 also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca2+ concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714–722; (1978) Thromb. Res.12, 619–628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E1 may initiate processes that decrease the Ca2+ concentration in the cytosol, so inhibiting both the Ca2+-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E1 did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca2+ concentration in the platelet cytosol. As prostaglandin E1 did inhibit the release of 5-hydroxy[3H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.
The effect of colchicine on human blood platelets under conditions of short-term incubation
Authors: Ribbi-Jaffe A , Apitz-Castro R
Published: August 2015

The effects of colchicine on ADP-induced aggregation and on the phosphorylation of tubulin-like protein from human blood platelets were studied. Colchicine at 2mM concentration completely inhibits ADP-induced aggregation after 8min incubation. Under the same inhibitory conditions, phosphorylation of tubulin-like materials in intact platelets was also impaired whereas the endogenous kinase activity of tubulin, isolated through polymerization–depolymerization cycles, was not affected. It was also shown that, under conditions of maximal inhibition of both aggregation and tubulin phosphorylation, colchicine does not penetrate into the cells. The results obtained suggest that the effect of colchicine on platelet aggregation might be mainly, although not exclusively, due to a non-specific effect of the alkaloid on the plasma membrane, rather than to a direct action of the drug on the microtubular protein subunits.
Cycloheximide as a probe of fibrinogen synthesis
Authors: Procyk R , Devlin T M , Ch'ih J J
Published: August 2015

Fibrinogen synthesis in the intact rat was perturbed by treatment with cycloheximide. Specific radioactivities of fibrinogen in plasma and liver both decreased at 2 h after treatment and increased over 2-fold by 18 h. Labelled-antibody–polyribosome binding experiments showed that more polyribosomes were engaged in fibrinogen synthesis at 18 h after treatment. Radioactivity of plasma fibrinogen chains from untreated control rats showed a constant ratio of A alpha–B beta/gamma = 1.03. At 2 h after cycloheximide treatment the A alpha- and B beta-chains showed the greatest decrease in labelling (A alpha–B beta/gamma = 0.66) and at 18 h all chains were much more labelled (the A alpha–B beta/gamma ratio chainged to 1.39). The observed imbalance in fibrinogen-chain synthesis suggests that cycloheximide has a selective effect on gene expression.
The turnover of cytochrome c in different skeletal-muscle fibre types of the rat
Authors: Terjung R L
Published: August 2015

The turnover of cytochrome c was determined in the three skeletal-muscle fibre types of adult male rats by a kinetic analysis that followed the time course of cytochrome c content change. Confirming evidence was obtained with double-labelling studies using delta-aminolaevulinate. Cytochrome c turnover was most rapid in the low-oxidative fast-twitch white fibre [t1/2 (half-life) about 4 days], slowest in the high-oxidative fast-twitch red fibre (t1/2 9-10 days) and relatively rapid in the high-oxidative slow-twitch red fibre (t1/2 5-6 days). Thus cytochrome c turnover does not strictly conform to either the appearance (i.e. red or white) or the contractile characteristics (i.e. fast or slow) of the muscle fibres. The synthesis rates needed to maintain the corresponding cytochrome c concentrations, however, were similarly high in the two mitochondria-rich red fibre types. These data illustrate that both the synthesis and degradation processes are important in establishing the cytochrome c concentrations that distinguish the different skeletal-muscle fibre types.
Enhancement of rat brain tryptophan metabolism by chronic ethanol administration and possible involvement of decreased liver tryptophan pyrrolase activity
Authors: Badawy A A , Punjani N F , Evans M
Published: August 2015

1. Chronic ethanol administration enhances rat brain 5-hydroxytryptamine synthesis by increasing the availability of circulating tryptophan to the brain. This increased availability is not insulin-mediated or lipolysis-dependent. 2. Under these conditions, tryptophan accumulates in the liver and apo-(tryptophan pyrrolase) activity is completely abolished, but could be restored by administration of regenerators of liver NAD+ and/or NADP+. 3. All four regenerators used (fructose, Methylene Blue, phenazine methosulphate and sodium pyruvate) prevented the ethanol-induced increase in liver tryptophan concentration and the increased availability of tryptophan to the brain. 4. It is suggested that the enhancement of brain tryptophan metabolism by chronic ethanol administration is caused by the decreased hepatic tryptophan pyrrolase activity. The results are briefly discussed in relation to previous work with ethanol. 5. Fructose enhances the conversion of tryptophan into 5-hydroxyindol-3-ylacetic acid in brains of ethanol-treated rats, whereas Methylene Blue inhibits this conversion in both control and ethanol-treated animals.
Polyprenyl phosphate sugars synthesized during slime-polysaccharide production by membranes of the root-cap cells of maize (Zea mays)
Authors: Green J R , Northcote D H
Published: August 2015

Two types of experiments were carried out; either maize roots were incubated in L-[1-3H]fucose or membranes were prepared from root tips and these were incubated with GDP-L-[U-14C]fucose or UDP-D-[U-4C]glucose. The radioactively labelled lipids that were synthesized in vivo and in vitro were extracted and separated into polar and neutral components. The polar lipids had the characteristics of polyprenyl phosphate and diphosphate fucose or glucose derivatives, and the neutral lipids of sterol glycosides (fucose or glucose). A partial separation of the glycolipid synthetase reactions was achieved. Membranes were fractionated into material that sedimented at 20,000g and 100,000g. Most of the polar glycolipid synthetase activity (for the incorporation of both fucose and glucose) was located in the 100,000 g pellet, and this activity was probably located in the endoplasmic reticulum. The neutral lipid, which contained fucose, was synthesized mainly by membranes of the 20,000g pellet, and the activity was probably associated with the dictyosomes, whereas the neutral glucolipids were synthesized by all the membrane fractions. It is suggested that the polar (polyprenyl) lipids labelled with fucose could act as possible intermediates during the synthesis of the glycoproteins and slime in the root tip.
Pools and protein synthesis in mammalian cells
Authors: Robertson J H , Wheatley D N
Published: August 2015

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.
Mass-spectral identification and purification of phycoerythrobilin and phycocyanobilin
Authors: Fu E , Friedman L , Siegelman H W
Published: August 2015

The bile-pigment chromophores of C-phycoerythrin (phycoerythrobilin) and C-phycocyanin (phycocyanobilin) were cleaved from their respective proteins with boiling methanol or butan-1-ol. They were purified as dicarboxylic acids by preparative reverse-phase liquid chromatography. Each pigment existed in two principal forms, which were characterized by using 20 pmol samples by proton-transfer chemical-ionization mass spectroscopy. These two principal forms were isomeric species, and all had protonated parent molecular ions with m/e 587, corresponding to a molecular weight of 586.
Differences in the degradation of native collagen by two microbial collagenases
Authors: Lecroisey A , Keil B
Published: August 2015

The early stages of degradation of native collagen by two bacterial collagenases were studied by electron microscopy and by automatic Edman degradation. The purified collagenase from Clostridium histolyticum was shown to cleave native collagen at several sites, but not progressively from the N-terminus, as had been previously suggested. The homogeneous collagenase from Achromobacter iophagus cleaves native collagen preferentially at two sites corresponding to the interbands 33-34 and 41-42. The latter lies within the region cleaved by the eukaryotic collagenases.
The inhibition of staphylococcal β-lactamase by clavulanic acid
Authors: Reading C , Hepburn P
Published: August 2015

Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.
Complete amino acid sequences of the three collagen-like regions present in subcomponent C1q of the first component of human complement
Authors: Reid K B M
Published: August 2015

The sequences of amino acid residues 38–51 of the A-chain, and residues 42–90 of the C-chain, of human subcomponent C1q are given. These results, along with previously published sequence data [Reid (1974) Biochem.J. 141, 189–203; Reid (1977) Biochem.J. 161, 247–251; Reid & Thompson (1978) Biochem.J. 173, 863–868] allow the presentation, and comparison with each other, of the complete amino acid sequences of the collagen-like regions found in the A-, B- and C-chains of human subcomponent C1q. Each chain has the continuity of its collagen-like Gly-X-Y repeating triplet amino acid sequence broken. The B- and C-chains have alanine residues at positions B-9 and C-36 where glycine might be expected. The A-chain has a threonine residue at position A-39, which is located between two Gly-X-Y triplets.
Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion
Authors: Gullick W J , Herries D G , Wood E J
Published: August 2015

The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 × 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the ‘collar’ of the molecule, and an aggregate some 20–30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000–300,000.
The determination of binding parameters when the total and free substrate concentrations are not approximately equal
Authors: Gains N
Published: August 2015

By using a standard graphical method values of Km and V may be found that are independent of the conditions and assumptions that the total substrate concentration approximates to its free concentration and that Km is much larger than the enzyme concentration. The procedure is also applicable to the determination of equilibrium binding parameters of a ligand to a macromolecule.
Effects of protein-deprivation on the regeneration of rat liver after partial hepatectomy
Authors: McGowan J , Atryzek V , Fausto N
Published: August 2015

Rats maintained on a protein-free diet for 3 days have an altered time course of hepatic DNA synthesis during liver regeneration. The delay in DNA synthesis is eliminated by the administration of casein hydrolysate (given as late as 6h after partial hepatectomy), but not by glucose or incomplete amino acid mixtures. Despite the change in the timing of DNA synthesis, the increases in hepatic amino acid pools, which take place at the earliest stages of the regenerative process, occur in a normal pattern in the regenerating liver of rats fed the protein-free diet. Protein-deprived rats have increased protein synthesis and decreased rates of protein degradation in the liver in response to partial hepatectomy, but these adaptations do not prevent a lag in protein accumulation and low protein/RNA ratios. The regenerating livers of these animals show a deficit in the accumulation of cytoplasmic polyadenylated mRNA as well as a smaller proportion of free polyribosomes. It is suggested that the deficit in free polyribosomes found in the regenerating liver of protein-deprived rats might be a consequence of the slow accumulation of mRNA species coding for intracellular proteins.