bluuebunny/crossref_metadata_2025 huggingface dataset:
Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R
Authors: Hood K , Bayley P M , Roberts G C
Published: August 2015

Circular-dichroism spectra (200–450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate–NADPH–enzyme ternary complex a! “couplet” c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.
Purification and properties of glutathione peroxidase from human placenta
Authors: Awasthi Y C , Dao D D , Lal A K , Srivastava S K
Published: August 2015

Glutathione peroxidase (glutathione–H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.
Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents
Authors: Newby Andrew C. , Chrambach Andreas
Published: August 2015

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.
Purification and some properties of a protein binding and deacylating initiator transfer ribonucleic acid
Authors: Pohlreich Petr , Kříž Oldřich , Tuháčková Zdena , Dušek Zdeněk , Hradec Jan
Published: August 2015

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNAfMet and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNAMet or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8–10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNAfMet is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNAfMet and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1–2mm). The ternary complex of the factor with methionyl-tRNAfMet and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNAfMet not required for translation.
Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens
Authors: Shinkai H , Yonemasu K
Published: August 2015

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388–1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.
Persistence of methylated bases in ribonucleic acid of syrian golden hamster liver after administration of dimethylnitrosamine
Authors: Margison G P , Margison J M , Montesano R
Published: August 2015

1. Syrian golden hamster liver ribosomal RNA was isolated up to 96 h after administration of [14C]dimethylnitrosamine at 25 mg/kg or 2.5 mg/kg body weight. 2. The chemical alkyation products, 7-methylguanine, 3-methylcytosine, O6-methylguanosine and 1-methyladenosine, were measured after acidic or enzymic hydrolysis of the RNA to bases or mononucleosides followed by ion-exchange chromatography. 3. Between 7 and 96 h, the relative amounts of alkylation products did not change with time even though the absolute amounts fell by approx. 80% and 51% after the high and low doses respectively. 4. The results suggest that base specific excision repair does not exist for RNA alkylation products in this experimental system.
Computed resolution and relative specific radioactivities of radiolabelled proteins synthesized by isolated gastric mucosal cells
Authors: Trotman Clive N. A. , Greenwell John R.
Published: August 2015

1. By radiolabelling, polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography, more than 50 soluble proteins in the molecular-weight range 15000–100000 were shown to continue to be synthesized after cells had been isolated from rat gastric mucosa. 2. Densitometric measurements of stained gels and fluorographic films were processed by computer to resolve individual overlapping Gaussian peaks corresponding to the protein bands. 3. Comparison of resolved peak areas of radioactivity and staining showed certain bands to have characteristically high relative specific radioactivities. 4. The computer programs (in FORTRAN) permit the analysis of a single densitometric trace or the simultaneous comparison of a corresponding pair of densitometric records of stained gels, or of fluorographic films, or a combination. Central processing unit time is used economically. 5. The programs identify the Gaussian components that contribute to the records and estimate their means, standard deviations and enclosed areas. These estimates are improved by a piecewise iterative method that minimizes the errors between the calculated and the experimental data. 6. Relative specific radioactivities are calculated as the normalized ratio of the area of a fluorographic film peak and the area of the corresponding stained gel peak. The computer programs have been deposited as Supplementary Publication SUP 50094 (55 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1978) 169, 5.
Changes in the contents of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle
Authors: Rowan Andrew N. , Newsholme Eric A.
Published: August 2015

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O2 uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and α-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD+/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.
Developmental pattern of rat intestinal brush-border enzymic proteins along the villus–crypt axis
Authors: Simon Patricia M. , Kedinger Michèle , Raul Francis , Grenier Jacques F. , Haffen Katy
Published: August 2015

At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase–isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase–isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase–isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus–crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific developmental pattern.
Demonstration of two functionally heterogenous groups within the activities of UDP-glucuronosyltransferase towards a series of 4-alkyl-substituted phenols
Authors: Wishart G J , Campbell M T
Published: August 2015

1. A simple colorimetric assay for UDP-glucuronosyltransferase activities towards phenolic substrates, using Folin & Ciocalteu's phenol reagent, is described. The assay is used to measure rat liver transferase activities towards substrates from a series of 4-alkyl-substituted phenols. 2. Activities towards phenol, 4-methylphenol and 4-ethylphenol develop near-adult values before birth, are precociously stimulated by dexa methasone in utero and are stimulated 3–4-fold by 3-methylcholanthrene in adult liver. These are assigned to a “late-foetal”! group of transferase activities. 3. Activities towards 4-n-propylphenol, 4-s-butylphenol and 4-t-butylphenol are negligible in late-foetal liver, developing to near-adult values in the first 4 postnatal days, and are not affected by dexamethasone or 3-methylcholanthrene. They are assigned to a “neonatal” group of transferase activities. 4. Although 4-ethylphenol and 4-n-propylphenol differ only by a single –CH2– moiety, this is sufficient to change the acceptability of these substrates respectively from the late-foetal to the neonatal group of transferase activities. The change is distinct, with no overlapping of substrate acceptability between the two groups of transferase activities. 5. From consideration of the above and other substrates, the two groups of transferase activities do not distinguish substrates on the basis of their molecular weights or lipophilicity. The distinguishing feature appears to be the specific molecular configurations of the substrates.
Fatty acid stimulation of membrane phosphatidylinositol hydrolysis by brain phosphatidylinositol phosphodiesterase
Authors: Irvine R F , Letcher A J , Dawson R M
Published: August 2015

The hydrolysis of membrane-bound phosphatidylinositol in rat liver microsomal fraction by the soluble phosphatidylinositol phosphodiesterase from rat brain was markedly stimulated by oleic acid or arachidonic acid. The stimulation did not require added calcium, although it was abolished by EDTA. Lysophosphatidylcholine also totally suppressed the stimulation. A possible role for the fatty acid content of a membrane in controlling phosphatidylinositol turnover is suggested.
No strict coupling of vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone)-dependent carboxylation and vitamin K1 epoxidation in detergent-solubilized microsomal fractions from rat liver
Authors: Wallin R
Published: August 2015

NAD(P)H dehydrogenase (‘DT-diaphorase’, EC 1.6.99.2) and vitamin K epoxidase were removed by affinity chromatography from detergent-solubilized microsomal fractions. Thereby the microsomal fractions normally carrying out vitamin K1-dependent carboxylation of the microsomal precursor proteins of the prothrombin complex were inactivated. Purified NAD(P)H dehydrogenase added to this system restored carboxylation in the presence of vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone) plus NADH. Vitamin K1 hydroquinone (2-methyl-3-phytyl-1,4-naphthoquinol) had no effect, in contrast with its effect in the intact system, where it can substitute for vitamin K1 plus NADH. The ability of NAD(P)H dehydrogenase to restore carboxylation in a system without vitamin K epoxidase activity shows that there is no obligatory coupling of the vitamin K1-dependent carboxylation with vitamin K1 epoxidation. These results suggest that the form of vitamin K1 that is active in the carboxylation reaction can be produced independently in two reactions: by NAD(P)H dehydrogenase in the reduction of the quinone and by vitamin K epoxidase in the epoxidation of the hydroquinone.
Differential effect of clofibrate on inflammation-induced alterations in plasma proteins in the rat
Authors: Powanda Michael C. , Abeles Fred B. , Bostian Karen A. , Fowler John P. , Hauer Edward C.
Published: August 2015

Daily intramuscular injections of clofibrate begun 6h before the initiation of inflammation induced by the subcutaneous injection of turpentine exerted a differential, dose-dependent inhibition of the anticipated acute-phase globulin response. Specifically, clofibrate at 140mg/kg muted the increase in α2-macrofoetoprotein, but did not affect that of seromucoid or haptoglobin and only transiently inhibited the rise in copper and the rebound in transferrin. A higher dose, 280mg/kg, markedly suppressed α2-macrofoetoprotein appearance and the rebound in transferrin, somewhat inhibited the increase in seromucoid and haptoglobin and only transiently affected the rise in plasma copper; 420mg of clofibrate/kg very nearly abolished the appearance of α2-macrofoetoprotein, markedly suppressed the transferrin rebound and the increases in seromucoid and haptoglobin and again only transiently affected the increase in copper. Clofibrate did not diminish the localized inflammatory response, did not cause microscopically detectable liver damage and did not prevent the hypozincaemia, hypoalbuminaemia and enhanced amino acid uptake by liver usually associated with inflammation. Thus it is unlikely that clofibrate exerted its dose-dependent selective inhibition by muting the initial stimulus or by impairing hepatic metabolism. This seemingly selective action of clofibrate on plasma-protein alterations during inflammation may provide a means of elucidating the function of individual acute-phase globulin during disease. Clofibrate of itself, apart from inflammation, produced decreases in plasma zinc, copper, transferrin and seromucoid and an increase in hepatic amino acid uptake that were to some extent dependent on the dose of the drug.
Characterization of prolactin binding by membrane preparations from rat liver
Authors: Silverstein A M , Richards J F
Published: August 2015

Binding sites for prolactin were identified in a plasma-membrane-enriched fraction isolated from livers of mature female rats. 125I-labelled sheep prolactin prepared by the lactoperoxidase procedure retained the same molecular integrity and binding affinity as the native hormone at physiological pH. The receptors bound prolactin from different species, whereas non-lactogenic hormones were not bound. The binding of 125I-labelled sheep prolactin was activated equally by bivalent and univalent cations, bivalent cations exerting their maximal effect at much lower concentrations. The association of 125I-labelled sheep prolactin with the receptor was a time- and temperature-dependent process. Partial dissociation was detected. The binding of 125I-labelled sheep prolactin was strongly influenced by pH, with an optimum observed at pH 6.5. Receptor activity was destroyed by Pronase and phospholipase C, whereas neuraminidase increased binding. Treatment of the membranes by ribonuclease and deoxyribonuclease did not affect the binding. Binding of 125I-labelled sheep prolactin was inhibited by p-chloromercuribenzoic acid, dithiothreitol and by brief exposure to high temperatures. Scatchard analysis of the binding of 125I-labelled sheep prolactin to receptors indicated that prolactin has a high affinity for its receptor. Binding of prolactin to liver membranes showed some properties different from those observed with mammary cells. Binding by these tissues differed in pH optimum, in effects of ions, and in response to neuraminidase.
Binding of ethidium bromide and quinacrine hydrochloride to nucleic acids and reconstituted nucleohistones
Authors: Chitre A. V. , Korgaonkar K. S.
Published: August 2015

Studies of binding of ethidium bromide and quinacrine hydrochloride to native DNA at low ionic strength indicate that for both compounds the binding is selective, with about one binding site for about four nucleotides. Annealing of unfractionated histones to DNA by a salt-gradient dialysis method slightly decreases the binding of the dyes to DNA. Similar observations made with reconstituted preparations by using individual histone fractions reveal that the arginine-rich histones (histones H3 and H4) are most effective in decreasing the binding. The binding studies with ethidium bromide at high ionic strength and with denatured DNA show that strong dye binding to DNA is strongly dependent on the ionic strength and on the secondary structure of DNA. The histones are not effective in decreasing the dye binding under conditions of high ionic strength. The results are consistent with the observations [Oliver & Chalkley (1974) Biochemistry13, 5093–5098; Axel, Melchoir, Sollner-Web & Felsenfield (1974) Proc. Natl. Acad. Sci. U.S.A.71, 4101–4105] that histones form some kind of surface structures on DNA through non-specific interactions and [Kornberg & Thomas (1974) Science184, 865–868; Kornberg (1974) Science184, 868–871; D'Anna & Isenberg (1974) Biochemistry13, 4992–4997; Vandegrift, Serra, Marve & Wagner (1974) Biochemistry13, 5087–5092] that the tendency of arginine-rich histones to aggregate may be an important factor in determining the structure of chromatin.
The catalytic activity of pig pepsin C towards small synthetic substrates
Authors: Auffret C A , Ryle A P
Published: August 2015

A series of small peptides has been synthesized and used to investigate the activity of a minor pig pepsin, pepsin C (EC 3.4.23.3). The peptides had the general formula A-Leu-Val-His-B. B was either OMe, NH2 or OH. With B = NH2 hydrolysis (kcat./Km) at 37 degrees C and pH 2.07 increased as A was Ac-Ala, Ac-Tyr, Ac-Phe and Ac-Ala-Phe. The pH dependence of the hydrolysis of Ac-Phe-Leu-Val-His-NH2 indicated the apparent pKa values of two catalytically important groups on the enzyme as 1.42 and 4.88. Inhibition of the hydrolysis of the same peptide by Ac-Phe at pH 3.01 showed a form of mixed non-competitive inhibition. Hydrolysis of Ac-Tyr-Leu-Val-His-OMe and the corresponding amide showed non-classical kinetics, which are discussed in terms of a substrate-activating mechanism. The results are discussed with reference to observations made by other workers on pig pepsin A.
The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A
Authors: Easterbrook-Smith Simon B. , Campbell Anne J. , Keech D. Bruce , Wallace John C.
Published: August 2015

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme– carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.
Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma
Authors: Ostlund-Lindqvist A M
Published: August 2015

Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk.
Effect of fixation time on measurement of 2′-deoxyribonucleoside 5′-triphosphate pools in the rat embyro
Authors: Ritter E J , Scott W J , Bruce L M
Published: August 2015

Values for the concentrations of deoxyribonucleoside triphosphates in rat embryos on day 12 of gestation, determined by high-pressure liquid chromatography, were artifactually two to three times as high in embryos fixed by cooling in ice/water followed by freezing on solid CO2, in 20s, as in those more rapidly/fixed in liquid N2, in 1 s.
The low availability of dietary choline for the nutrition of the sheep
Authors: Neill A R , Grime D W , Snoswell A M , Northrop A J , Lindsay D B , Dawson R M C
Published: August 2015

1. Choline, which is present in the diet of the sheep either in the non-esterified form or combined in phospholipids, is rapidly degraded in the rumen. The ultimate product formed from the N-methyl groups is methane. 2. Analysis of the non-esterified choline and the phosphatidylcholine in ruminal and abomasal digesta indicate that the phospholipid is the main vehicle for the passage of choline to the lower digestive tract. 3. The concentration of phosphatidylcholine in abomasal digesta is lower than that of ruminal digesta, which is in line with a selective retention of protozoa in the rumen as observed by others. 4. On defaunation of the rumen to remove ciliated protozoa the concentration of phosphatidylcholine in ruminal digesta falls markedly and becomes lower than that in abomasal digesta. 5. Calculation shows that the adult sheep obtains at most only about 20–25 mg of effective choline per day from its diet (0.002–0.0025% of dietary total dry-weight intake). This is some fifty times less than the minimum required to avoid pathological lesions and death in other species investigated (0.1%+ of dietary dry-weight intake). 6. Sheep liver can synthesize choline from [14C]ethanolamine both in vitro and in vivo, but the synthesis of choline per kg body weight is many times less than it is in the rat. 7. The intact sheep oxidizes an injected dose of [1,2-14C]choline to CO2 at a rate that is several times less than that observed for the rat. This could help to explain the apparent minimal requirement of sheep for dietary choline.
Effects of some chlorinated sugar derivatives on the hexose transport system of the blood/brain barrier
Authors: Cremer J E , Cunningham V J
Published: August 2015

The inhibition of D-glucose transport into brain by several hexose analogues has been investigated in adult anaesthetized rats. D-Glucose was transported with apparent Vmax. = 1.22 mumol/g per min, Km = 11.12 mM and Kd = 0.008 ml/g per min. 6-Chloro-6-deoxyglucose was transported with corresponding values of Vmax. = 1.33 mumol/g per min, Km = 5.5 mM and Kd = 0.0155 ml/g per min and inhibited D-glucose transport with apparent Ki = 3.01 mM. 6-Chloro-6-deoxymannose, 6-chloro-6-deoxygalactose and 6-tosyl-6-deoxygalactose also inhibited D-glucose transport, but 6-chloro-6-deoxyfructose was without effect. The results were consistent with a model for glucose transport at the blood/brain interface that involves a hydrophobic site on the transport protein at or near the 6-position of bound glucose.
Inhibition of rat liver mevalonate pyrophosphate decarboxylase and mevalonate phosphate kinase by phenyl and phenolic compounds
Authors: Shama Bhat C , Ramasarma T
Published: August 2015

1. Mevalonate pyrophosphate decarboxylase of rat liver is inhibited by various phenyl and phenolic acids. 2. Some of the phenyl and phenolic acids also inhibited mevalonate phosphate kinase. 3. Compounds with the phenyl-vinyl structure were more effective. 4. Kinetic studies showed that some of the phenolic acids compete with the substrates, mevalonate 5-phosphate and mevalonate 5-pyrophosphate, whereas others inhibit umcompetitively. 5. Dihydroxyphenyl and trihydroxyphenyl compounds and p-chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. 6. Of the three mevalonate-metabolizing enzymes, mevalonate pyrophosphate decarboxylase has the lowest specific activity and is probably the rate-determining step in this part of the pathway.
A simple procedure for the isolation of a major amelogenin polypeptide component
Authors: Fincham A G
Published: August 2015

A simple, reproducible gel-filtration procedure for the isolation of one of the major ‘J-Group’ polypeptides of the bovine foetal dental-enamel matrix is described. The purified polypeptide was characterized by amino acid analysis, electrophoresis, cleavage with CNBr and N-terminal analyses. The isolated component is shown to be closely similar to an amelogenin component described by other workers.
Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent
Authors: Birnbaumer M E , Schrader W T , O'Malley B W
Published: August 2015

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.
A comment on the ‘jack-knife’ technique for analysing enzyme-kinetic data
Authors: Duggleby R G
Published: August 2015

The use of the ‘jack-knife’ technique in the analysis of enzyme-kinetic data [Cornish-Bowden & Wong (1978] Biochem. J. 175, 969–976) is examined. The method can give parameter estimates that appear to be incorrect.
Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions
Authors: Russell Nicholas J. , Harwood John L.
Published: August 2015

The acyl lipids and their constituent fatty acids were studied in the photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides, which were grown under photosynthetic and non-photosynthetic conditions. The major lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in each bacterium. The two Rhodopseudomonas species also contained significant quantities of phosphatidylcholine. Other acyl lipids accounted for less than 10% of the total. On changing growth conditions from non-photosynthetic to photosynthetic a large increase in the relative proportion of phosphatidylglycerol was seen at the expense of phosphatidyl-ethanolamine. In Rhodospirillum rubrum the fatty acids of the major phospholipids showed an increase in the proportion of palmitate and stearate and a decrease in palmitoleate and vaccenate on changing growth conditions to photosynthetic. In contrast, the exceptionally high levels (>80%) of vaccenate in individual phospholipids of Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides were unaffected by changing growth conditions to photosynthetic. Analysis of the lipids of chromatophores, isolated from the three bacteria, showed that these preparations were enriched in phosphatidylglycerol. The large increase in this phospholipid, seen during growth under photosynthetic conditions, appeared, therefore, to be due to a proliferation of chromatophore membranes. Possible roles for acyl lipids in the formation and function of the photosynthetic apparatus of bacteria are discussed.
1H nuclear-magnetic-resonance spectra of the methyl group of the acetamido moiety and the structure of acid glycosaminoglycans in solution
Authors: Scott J E , Heatley F
Published: August 2015

The 1H resonances of the methyl group in the acetamido moiety of several types of glycosaminoglycans are reported at 300 MHz in 2H2O. Dermatan sulphates with various L-iduronate/D-glucuronate ratios are compared with chrondroitin sulphates with various contents and positions of substitution of sulphate esters. Hyaluronate oligomers are compared with 2-acetamido-2-deoxy-D-glucose, and with heparan sulphate and keratan sulphate. The major determinant of the chemical shift of the acetamido methyl resonance is the closeness of approach between carboxy groups and the acetamido group, in agreement with chemical evidence based on periodate-oxidation kinetics.
Tissue-culture Cell fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients
Authors: Graham J M , Coffey K H M
Published: August 2015

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.
Phosphatidylinositol metabolism in rat hepatocytes stimulated by glycogenolytic hormones. Effects of angiotensin, vasopressin, adrenaline, ionophore A23187 and calcium-ion deprivation
Authors: Billah M. Motassim , Michell Robert H.
Published: August 2015

1. The effects on phosphatidylinositol metabolism of three Ca2+-mobilizing glycogenolytic hormones, namely angiotensin, vasopressin and adrenaline, have been investigated by using rat hepatocytes. 2. All three hormones stimulate both phosphatidylinositol breakdown and the labelling of this lipid with 32P. 3. The response to angiotensin occurs quickly, requires a high concentration of the hormone and is prevented by [1-sarcosine, 8-isoleucine]angiotensin, a specific angiotensin antagonist that does not prevent the responses to vasopressin and to adrenaline. This response therefore seems to be mediated by angiotensin-specific receptors. 4. [1-Deaminocysteine,2-phenylalanine,7-(3,4-didehydroproline),8-arginine] vasopressin, a vasopressin analogue with enhanced antidiuretic potency, is relatively ineffective at stimulating phosphatidylinositol metabolism. This suggests that the hepatic vasopressin receptors that stimulate phosphatidylinositol breakdown are different in their ligand selectivity from the antidiuretic vasopressin receptors that activate renal adenylate cyclase. 5. Incubation of hepatocytes with ionophore A23187, a bivalent-cation ionophore, neither mimicked nor appreciably changed the effects of vasopressin on phosphatidylinositol metabolism, suggesting that phosphatidylinositol breakdown is not controlled by changes in the cytosol Ca2+ concentration. This conclusion was supported by the observation that hormonal stimulation of phosphatidylinositol breakdown and resynthesis persists in cells incubated for a substantial period in EGTA, although this treatment somewhat decreased the phosphatidylinositol response of the hepatocyte. The phosphatidylinositol response of the hepatocyte therefore appears not to be controlled by changes in cytosol [Ca2+], despite the fact that this ion is thought to be the second messenger by which the same hormones control glycogenolysis. 6. These results may be an indication that phosphatidylinositol breakdown is an integral reaction in the stimulus–response coupling sequence(s) that link(s) activation of α-adrenergic, vasopressin and angiotensin receptors to mobilization of Ca2+ in the rat hepatocyte.
Purification of the membrane-bound hydrogenase of Escherichia coli
Authors: Adams M W W , Hall D O
Published: August 2015

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.
Inhibition of bacteriophage-Q β ribonucleic acid polymerase by guanosine triphosphate analogues
Authors: Brooks R R
Published: August 2015

Sixteen compounds related to GTP were evaluated as inhibitors of bacteriophage-Q beta poly(C)-dependent poly(G) polymerase. Non-phosphorylated compounds, including guanine, guanosine and deoxyguanosine, were inactive. Phosphorylated compounds gave significant inhibition at millimolar concentrations. For nucleotides the feature important for inhibition was the 5′-phosphate chain. Four triphosphates, XTP, ITP, 7-methyl-GTP and 2′-O-methyl-GTP, gave 50% inhibition of both the poly(C)- and poly(U2,C)-dependent reactions at concentrations from 0.1 to 5 mM. XTP was 10-fold more potent an inhibitor of the reaction with poly(U2,C) as template. None of these four compounds was able to substitute for GTP as substrate to a significant extent. The most active compound, 2′-O-methyl-GTP, was a competitive inhibitor (Ki = 0.4 mM) of GTP in the poly(C)-dependent reaction.
Microbubbles in replicating nuclear deoxyribonucleic acid from Physarum polycephalum
Authors: Gillespie D A F , Hardman N
Published: August 2015

Clusters of microbubbles, represent probable sites of newly initiated DNA synthesis, were identified in nuclear DNA from Physarum polycephalum by using the electron microscope. Their presence is associated specifically with S-phase. Each microbubble corresponds in size to a replicating segment of DNA about 100-5000 nucleotide residues in length. The DNA structures containing microbubbles are metastable, and revert to native DNA in the presence of moderate concentrations of formamide used to prepare samples for electron microscopy. It is suggested that each cluster of microbubbles may correspond to a unit of replication (a replicon) in Physarum DNA.
A new series of long-chain dicarboxylic acids with vicinal dimethyl branching found as major components of the lipids of Butyrivibrio spp
Authors: Klein R A , Hazlewood G P , Kemp P , Dawson R M C
Published: August 2015

1. Some members of the genus Butyrivibrio, including a general fatty acid auxotroph (strain S2), contain as a major part of their complex lipids a high-molecular-weight component that is probably formed by the union of two fatty acid chains [Hazlewood & Dawson (1979) J. Gen. Microbiol. 112, 15–27]. 2. Proton and 13C n.m.r. and i.r. and mass spectroscopy were used to examine a homologous series of these moieties and, in addition, the hydrocarbon derivative of one homologue and several synthetic compounds. 3. The results indicate that the high-molecular-weight components are a series of long-chain dicarboxylic acids containing vicinal dimethyl branching, located near the centre of the chain.
2,2′-Dithiodipyridine activates aldehyde dehydrogenase and protects the enzyme against inactivation by disulfiram
Authors: Kitson T M
Published: August 2015

Small concentrations of 2,2′-dithiodipyridine cause a rapid activation of sheep liver cytoplasmic aldehyde dehydrogenase in the presence of NAD+. Enzyme pre-modified by 2,2′-dithiodipyridine is largely protected against the potent inactivatory effect of disulfiram. 2,2′-Dithiobis-(5-nitropyridine) inactivates the enzyme. The implications of these results are discussed with reference to various possible classes of thiol group in aldehyde dehydrogenase.
β-Galactoside-binding muscle lectins of man and monkey show antigenic cross-reactions with those of bovine origin
Authors: Childs R A , Feizi T
Published: August 2015

Endogenous beta-galactoside-binding lectins were isolated from human heart and from human and rhesus-monkey skeletal muscles. Gel precipitation and radioimmunoassays with rabbit antisera to calf heart lectin revealed antigenic cross-reactions between the primate and bovine muscle lectins.
Synthesis of cytoplasmic membrane during growth and division of Escherichia coli. Dispersive behaviour of respiratory nitrate reductase
Authors: Cadenas E , Garland P B
Published: August 2015

We have used the penicillin selection method of Autissier & Kepes [(1972) Biochimie 54, 93–101] to study the segregation of membrane-bound respiratory nitrate reductase (EC 1.9.6.1) in Escherichia coli for the three generations after cessation of nitrate reductase synthesis caused by withdrawal of nitrate from the growth medium. We also included a physical separation procedure that permitted direct assay for nitrate reductase activity among all fractions produced by the penicillin selection method. We conclude that the segregation of nitrate reductase after cell division is dispersive, and not semi-conservative as proposed by Autissier & Kepes (1972).