bluuebunny/crossref_metadata_2025 huggingface dataset:
Location of O-methyl sugars in antigenic (lipo-)polysaccharides of photosynthetic bacteria and cyanobacteria
Authors: Tharanathan R N , Mayer H
Published: August 2015

An attempt was made to localize a number of O-methyl sugars in lipopolysaccharides and antigenic polysaccharides isolated from photosynthetic bacteria and from cyano-bacteria. Methylation analysis with [2H3]methyl iodide as methylating agent was the method of choice. One has to differentiate between (lipo-)polysaccharides having only trace amounts (less than 1% of polymer dry weight) of O-methyl sugars and those having them in larger amounts (more than 4% of polymer dry weight). In the former case O-methyl sugars occupy either non-reducing or reducing terminals. When present in larger quantities they may be present as part of each repeating unit either in chain-linked or in terminal positions or in both. A possible role of O-methyl sugars in biosynthesis of O-chains, and their contribution to the lipophilic character of the cell surface are discussed.
The precise and entire antigenic structure of native lysozyme
Authors: Atassi M Z , Lee C L
Published: August 2015

The exact boundary, residue, conformational and directional definitions of the three antigenic sites of native hen's egg-white lysozyme are described. The results clearly reveal that the three antigenic sites account quantitatively for the total antigenic reactivity of the protein. Thus the entire antigenic structure of lysozyme has now been precisely determined and is briefly discussed here, together with the power of the surface-stimulation synthetic concept.
I-cell disease. A hypothesis for the structure of the carbohydrate recognition site on β-d-N-acetylhexosaminidase
Authors: Vladutiu G D
Published: August 2015

I-cell disease (mucolipidosis II) is presented as a model for endo- and exo-cytosis phenomena in man. A hypothesis is presented for the structure of the carbohydrate recognition site on fibroblast-derived beta-D-N-acetylhexosaminidase that may extend to the other affected hydrolases and that is responsible for specific uptake of the enzyme by fibroblasts. The proposed neuraminidase deficiency in I-cell disease is discussed in the light of its significance in influencing the final sugar sequence in the carbohydrate structure of the recognition site.
Chemical modification as a probe of conformational changes in transfer ribonucleic acid on aminoacylation
Authors: Lowdon M , Goddard J P
Published: August 2015

Treatment of Escherichia coli CA265 phenylalanyl-tRNA with 3M-NaHSO3, pH6.0, at 25 degrees C resulted in modification of four bases and in the deacylation of the charged tRNAphe. The similarity of the rates of base modification and of the deacylation of the phenylalanyl-tRNA permitted the isolation of partially modified phenylalanyl-tRNAphe and partially modified deacylated tRNAphe. The sites and extents of base modification in these fractions were determined and found to be the same as those in uncharged tRNAphe modified under identical conditions. These findings are discussed in relation to previous evidence for and against a conformational change in tRNA on its aminoacylation. The methods described should prove adaptable to study of other aminoacyl-tRNA species.
Phospholipid content and activity of pure uridine diphosphate-glucuronyltransferase from rat liver
Authors: Burchell B , Hallinan T
Published: August 2015

Rat liver phospholipids were radioactively labeled in vivo before purification of UDP-glucuronyltransferase to homogeneity. The pure enzyme contained very little phospholipid (approx. 0.7 mol of phospholipid/mol of protein). The solubilization detergent Lubrol 12A9 appeared to act as a phospholipid substitute, capable of supporting UDP-glucuronyltransferase activity. Phospholipase C did not inhibit the pure enzyme activity and pure UDP-glucuronyltransferase was stimulated by 40–100% by the addition of phospholipid dispersions.
All factors required for protein synthesis are retained on heparin bound to Sepharose
Authors: Hradec J , Dušek Z
Published: August 2015

1. Postmitochondrial supernatants of rabbit reticulocyte lysates were chromatographed on heparin bound to Sepharose 4B, and the fraction retained on affinity columns was separated by subsequent gel filtration on Sepharose 4B into three fractions, two of them active in protein synthesis. 2. The heavier fraction sedimented at 40S and contained more than 10% RNA. This consisted predominantly of a 12S component, with smaller amounts of the 9S and 4S RNA species. The lighter fraction (18-20S) was composed of proteins with less than 1% RNA. 3. Different enzymic activities were associated with these fractions. 4. In the presence of both fractions, efficient translation took place on combined ribosomal subunits of rat liver with added cofactors. Globin messenger ribonucleoprotein stimulated this translation 5-6-fold. 5. Relatively large complexes of all factors required for protein synthesis are apparently isolated from reticulocytes by affinity chromatography on heparin-Sepharose 4B. Such complexes may occur naturally in the cytoplasm of mammalian cells.
Biochemistry of metallocenes. Identification of the major metabolite of acetylruthenocene
Authors: Taylor A J , Wenzel M
Published: August 2015

1. After oral administration of acetylruthenocene to rats, metabolites were detected in bile and urine. 2. The major metabolite, which is present in both bile and urine, is a glucuronide with the structure: C5H5-Ru-C5H4-CO-CH2-O-C6H9O6 3. The metabolite was identified by mass spectrometry of the permethylated glucuronide and mass spectrometry and n.m.r. of the aglycone. 4. The nature of the metabolite is discussed, and a comparison is made with the metabolism of the benzene analogue, acetophenone.
Changes in the lipoprotein lipase (clearing-factor lipase) activity of white adipose tissue during development of the rat
Authors: Cryer A , Jones H M
Published: August 2015

The lipoprotein lipase (clearing-factor lipase) activity of the white adipose tissue from rats aged between 1 and 145 days was determined. Five adipose-tissue sites (epididymal, uterine, subcutaneous, perirenal and intramuscular) together with serum concentrations of triacylglycerol, cholesterol and glucose were studied. The pattern of enzyme-activity change was remarkably similar in all the sites studied, although the growth of the tissues proceeded non-uniformly. After a peak of activity early in suckling, lipoprotein lipase activity fell to low values by 20 days of age. At weaning (21 days) the activity increased sharply and within 5 days high values were regained. The serum triacylglycerol and cholesterol concentrations were low at birth and reached peaks of concentration coincidentally with the minima of white-adipose-tissue lipoprotein lipase activities, seen late in suckling. The changes in enzyme activity were related to other metabolic changes in adipose tissue and with the known changes in plasma insulin concentrations occurring during development.
Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours
Authors: Malkinson A M , McSwigan C E
Published: August 2015

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.
Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats
Authors: Siess E A , Brocks D G , Wieland O H
Published: August 2015

In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.
The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen
Authors: Greenwood C , Barber D , Parr S R , Antonini E , Brunori M , Colosimo A
Published: August 2015

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 × 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.
Thermodynamics for the interaction of ε-dinitrophenyl-l-lysine and bovine colostral anti-dinitrophenyl immunoglobulin G2
Authors: Mukkur T K S
Published: August 2015

The effect of varying the temperature over a wide range (4–60 degrees C) on the binding of epsilon-dinitrophenyl-L-lysine to bovine colostral anti-dinitrophenyl immunoglobulin G2 yielded a non-linear van′t Hoff plot. The extent of curvature was indicative of a large positive heat-capacity change, and the thermodynamic parameters, calculated by using a non-linear least squares computer procedure, revealed an enthalpy–entropy-compensation mechanism for hapten-antibody binding. The enthalpy factor was found to be the primary contributor for the complex-formation at low temperatures, but at increasing temperatures the entropy factor assumed greater importance. At physiological temperature (39 degrees C), the entropy factor was the major contributor to the free energy of reaction.
Properties of matrix-bound dimer and monomer derivatives of immobilized creatine kinase from rabbit skeletal muscle
Authors: Bickerstaff G F , Price N C
Published: August 2015

Dimeric creatine kinase (EC 2.7.3.2) from rabbit skeletal muscle can be immobilized via a single subunit to CNBr-activated Sepharose 4B and subsequently treated with guanidine hydrochloride followed by renaturation to yield a catalytically active matrix-bound subunit derivative. The importance of the intact dimeric structure in the activation of the enzyme by acetate was demonstrated. Immobilization did not appear to alter the pH optimum of the enzyme, and the kinetic parameters fot the matrix-bound derivatives were generally similar to those for the soluble enzyme, but the matrix-bound derivatives showed higher thermal stability and greater resistance to denaturation than did the soluble enzyme. The rates of reaction of thiol groups of the matrix-bound derivatives with iodoacetamide in the absence and in the presence of combinations of substrates were similar to those of the soluble enzyme. Studies with 5,5′-dithiobis-(2-nitrobenzoic acid) and with iodoacetamide revealed the presence of an additional reactive thiol group in the matrix-bound subunit derivative, which is presumably masked in the dimeric derivatives.
Histone composition of a chromatin fraction containing ribosomal deoxyribonucleic acid isolated from the macronucleus of Tetrahymena pyriformis
Authors: Jones R W
Published: August 2015

The histone compositions of a chromatin fraction containing ribosomal DNA and of the remaining macronuclear chromatin of Tetrahymena pyriformis was analysed by gel electrophoresis. These chromatin fractions were used as models for transcriptionally active and inactive chromatin respectively. The extent of histone modification, as indicated by the distribution of histone between differently charged subspecies in acid-urea gels, is not grossly different in the two chromatin fractions. However, histone H1 is present, but may be differently modified in the two chromatin fractions. The histone/DNA ratio in ribosomal chromatin, measured after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of samples of chromatin, was found to be the same whether chromatin was extracted from growing or stationary organisms, and to be approx. 40% of this ratio in the remaining macronuclear chromatin. The implications of these results for the possible structure of transcriptionally active chromatin are discussed.
Inactivation and modification of lactate oxidase with fluorodinitrobenzene
Authors: Soon C Y , Shepherd M G , Sullivan P A
Published: August 2015

1. Dinitrophenylation of 2 +/- 0.2mol of residues/mol of enzyme-bound FMN resulted in the complete inactivation of the flavoenzyme L-lactate oxidase. 2. Hydrolysates of the inactivated enzyme contained 1mol each of Nim-Dnp-histidine (abbreviation: Dnp-,2,4-dinitrophenyl-; Nim indicates that either of the N atoms in the imidazole ring is substituted) and epsilon-Dnp-lysine/mol of FMN. 3. Competitive inhibitors decreased the extent of inactivation to a 10% loss of activity, and dinitrophenylation was decreased from 2 to approx. 0.5mol/mol of FMN. Only Nim-Dnp-histidine was detected in the hydrolysates. 4. Although the dinitrophenylated enzyme did not possess enzyme activitiy, L-lactate reduced approx. 50% of the enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme formed a complex with bisulphite. 6. The modified enzyme (2mol of Dnp/mol of FMN) was unable to bind substrate analogues and competitive inhibitors.
Primary structure of the major β-chain of rat haemoglobins
Authors: Garrick Laura M. , Sloan Richard L. , Ryan Thomas W. , Klonowski Thomas J. , Garrick Michael D.
Published: August 2015

The amino acid sequence of the major β-chain, IIβ, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the ε-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 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 indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.
A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum
Authors: Christeller J T , Laing W A
Published: August 2015

The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25×10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7×10(-3)-10×10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11–18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase.
Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase
Authors: Lodola A , Parker D M , Jeck R , Holbrook J J
Published: August 2015

1. The pH-dependencies of the binding of NADH and reduced nicotinamide–benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3–6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.
Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase
Authors: Franzen J S , Marchetti P , Ishman R , Ashcom J
Published: August 2015

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.
Comparative studies on intrinsic factor and cobalophilin in different parts of the gastrointestinal tract of the pig
Authors: Marcoullis George , Merivuori Hannele , Gräsbeck Ralph
Published: August 2015

The vitamin B12 binders in the pig pyloric mucosa gastric and intestinal juice from the upper gastrointestinal tract were fractionated into only two molecular forms, classified as intrinsic factor and cobalophilin. The unsaturated vitamin B12-binding power due to cobalophilin was lower in the intestinal than in the gastric juice. Electrofocusing revealed that intrinsic factor and cobalophilin in the intestinal juice contained more of the ‘neutral’-type isoproteins, and the suggestion is made that this is due to enzyme activity. The gastric-juice intrinsic factor contained more acidic isoproteins, which supports the hypothesis that carbohydrate is added on to the polypeptide chain of this protein before it is secreted into gastric juice. The gastric- and intestinal-juice cobalophilins, studied also by electrofocusing, differed from that of pyloric mucosa and they appeared to be of salivary origin. With regard to molecular dimensions there was no significant difference between the intrinsic factors and cobalophilins from all sources studied. All cobalophilins had molecular weights by the formula of Svedberg of approx. 92500, Stokes radii of 4.62nm and sedimentation coefficients of 5.15S. The corresponding values for the intrinsic factors were 63600, 3.57nm and 4.38S. In addition, the intrinsic factors exhibited similar avidities for binding to the solubilized ileal intrinsic-factor receptor. Also the intrinsic factors and cobalophilins, irrespective of their source, bound to the analogous specific xenoantibodies with the same avidity. The present results demonstrate that intrinsic factor remains practically unaltered during its passage through the proximal intestine and render unlikely the speculations made about the presence of an endogenous binder for intrinsic factor as well as the existence of a ‘pancreatic intrinsic factor’. In addition, they are compatible with the theory that the interference by undegraded cobalophilin may be the reason for the abnormal vitamin B12 absorption observed in patients with pancreatic insufficiency.
Removal by transamination and scission of residues from the peptide representing the copper-transport site of serum albumin
Authors: Sarkar B , Dixon H B F , Webster D
Published: August 2015

The peptide Asp-Ala-His-NH-Me was subjected to removal of its N-terminal residue by transamination and scission. Despite the high affinity of the peptide for Cu2+ ions, they catalysed its transamination smoothly. Two main transamination products were found, a complication previously observed with another peptide with an N-terminal aspartic residue, but their scission gave a single product, Ala-His-NH-Me. This was subjected to a further cycle of transamination and scission, and gave a single product after each step. For scission of transaminated peptides it proved unnecessary to remove them from transamination reagents provided that transamination was stopped with EDTA before adding the scission reagent.
Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes
Authors: Werb Z , Dingle J T , Reynolds J J , Barrett A J
Published: August 2015

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.
A general method for purification of deoxycytidine kinase
Authors: Baxter A , Currie L M , Durham J P
Published: August 2015

Deoxycytidine kinase from a variety of animal tissues binds to Cibacron Blue 3G-A coupled to Sepharose CL-6B. The enzyme can be selectively eluted with ATP to yield single-step purifications of 17-89-fold. The affinity adsorbent is re-usable.
Effect of non-histone chromosomal proteins on transcription in vitro in sea-urchin
Authors: Di Mauro E , Pedone F , Pomponi M
Published: August 2015

Non-histone chromosomal proteins prepared from chromosomal material of the sea-urchin Paracentrotus lividus affect RNA synthesis in vitro. 1. The extent of transcription can be radically changed from inhibition to stimulation, depending on the DNA/non-histone chromosomal proteins ratio. 2. A correlation exists between stage of development and influence on transcription. 3. Non-histone chromosomal proteins exert their action by intervening directly on some initiation step of RNA synthesis, as shown by the numbers of initiation events that take place in their presence or absence. 4. Stimulatory activity is observed only in restrictive conditions of ionic strength and temperature. These observations are in agreement with models that predict for non-histone chromosomal proteins a regulatory role on the transcription process exerted through a modulation of promoter availability.
The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment
Authors: Dipple I , Houslay M D
Published: August 2015

1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The ‘break’ points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5′-Nucleotidase activity was stimulated by benzyl alcohol, and the ‘break’ point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.
The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential
Authors: Sorgato M C , Ferguson S J , Kell D B , John P
Published: August 2015

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0′ + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.
Developmental changes in the activity of lipoprotein lipase (clearing-factor lipase) in rat lung, cardiac muscle, skeletal muscle and brown adipose tissue
Authors: Cryer A , Jones H M
Published: August 2015

The lipoprotein lipase activity of the lung, skeletal muscle, heart muscle and brown adipose tissue of the rat was studied during the period from late foetal to adult life. The enzyme activity in all four tissues emerged substantially during the first 24th after birth. Subsequently, heart and lung enzyme activity remained relatively constant per unit wet weight of tissue. The enzyme activity present in brown adipose tissue and skeletal muscle was elevated per unit weight of tissue during suckling compared with other periods of life. Delivery of near-term foetuses stimulated the emergence of enzyme activity in all four tissues with the same time course as that evoked by normal delivery. The significance of the presence of the enzyme in the tissues and the activity changes which occurred during development are discussed in relation to possible mechanisms of control.
Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro
Authors: Cates G A , Holland P C
Published: August 2015

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5′-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.
Prostaglandin receptors on human platelets. Structure-activity relationships of stimulatory prostaglandins
Authors: MacIntyre D E , Salzman E W , Gordon J L
Published: August 2015

1. Synthetic analogues of prostaglandins E2 or F2a (monocyclic bisenoic prostaglandins), like the endogenous prostaglandin endoperoxides (prostaglandins G2 and H2) from platelets, and like synthetic analogues of prostaglandin H2 (bicyclic bisenoic prostaglandins), can induce aggregation of human platelets, although prostaglandins E2 and F2a themselves are inactive. 2. All the prostanoid compounds that induce platelet aggregation release 5-hydroxytryptamine from platelet dense bodies, but do not release beta-N-acetylglucosaminidase from lysosomal granules. Arachidonic acid evokes a similar response. 3. All endoperoxide analogues tested (bicyclic compounds) were powerful platelet stimulants, and all active compounds (whether mono- or bi-cyclid) apparently acted via the same receptor as the endogenous prostaglandin endoperoxides. 4. The nature and stereospecificity of substituents at positions 11 and 15 (or 16) on prostaglandin E2 are critical determinants for platelet-stimulating activity: deoxy substitution at position 11 plus methylation at position 15 (or 16) produces a potent stimulant, particularly if the groups around C-15 are in the S configuration. 5. The effects of these structural modifications are apparently due to, at least in part, a change in side-chain conformation.
Differentiation of the changes in alkaline phosphatase from calcium ion-activated adenosine triphosphatase activities associated with increased calcium absorption in chick intestine
Authors: Lane S M , Lawson D E M
Published: August 2015

The pattern of response of the intestinal enzymes Ca2+-activated adenosine triphosphatase and alkaline phosphatase in the chick to 1,25-dihydroxycholecalciferol is consistent with a role for the former but not the latter enzyme in the vitamin D-dependent absorption of calcium.
Dibutyryladenosine 3′:5′-cyclic monophosphate-mediated changes in rat cells involve macromolecular alterations in vinblastine-precipitable proteins
Authors: Meza A T , Rieber M
Published: August 2015

ts-NT3-KR rat cell cultures show the loss of three components in the molecular-weight region 200,000–250,000 when exposed to dibutyryl cyclic AMP, under conditions of both restriction and expression of the transformed phenotype. Vinblastine is able to precipitate preferentially from control cultures the species that are decreased by exposure to the cyclic nucleotide. Serum-starved cultures exposed to dibutyryl cyclic AMP reveal differences in their vinblastine precipitates, depending on whether the expression of the transformation phenotype is restricted or not.
Interference by branched-chain amino acids in the assay of alanine with alanine dehydrogenase
Authors: Lund P , Baverel G
Published: August 2015

Commercial preparations of alanine dehydrogenase from Bacillus subtilis are contaminated to varying extents with activity towards branched-chain amino acids. The Km values for these amino acids are of the same order as for L-alanine (about 10(-3)M). The branched-chain amino acid dehydrogenase activity is lost on dialysis for 2–4h against water or 2mM-EDTA.
The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei
Authors: MacGillivray A J , Johnston C , MacFarlane R , Rickwood D
Published: August 2015

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.
Isolation and properties of brain α-actinin
Authors: Schook W , Ores C , Puszkin S
Published: August 2015

alpha-Actinin isolated from bovine brain migrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis like muscle alpha-actinin with an apparent mol.wt. of 100000 and cross-reacted with antibodies to muscle alpha-actinin. Brain alpha-actinin modulated actin-myosin Mg2+-activated adenosine triphosphatase activity and, when bound by polystyrene particles, was found to bind muscle actin and tropomyosin from solution. Brain alpha-actinin, in conjunction with the other components of the contractile and relaxing complex, may play a role in the release of neurotransmitters from synaptic vesicles.
Isolation of the C-fragment and C′-fragment of lipotropin from pig pituitary and C-fragment from brain
Authors: Smyth D G , Snell C R , Massey D E
Published: August 2015

Three novel peptides derived from lipotropin, the C-Fragment (residues 61-91), C′-Fragment (61-87) and N-Fragment (1-38), were isolated from pig pituitary, and the C-Fragment was shown to be present in brain. The experimental procedures developed for their isolation are described. The formation of each of the fragments involves enzymic cleavage of lipotropin at consecutive basic residues, with specificity identical with that involved in the activation of known prohormones. In brain assays C-Fragment exhibits a range of biological activities related to its ability to act as an inhibitory neurotransmitter.
Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography
Authors: Fifis T , Scopes R K
Published: August 2015

1. Affinity elution chromatography was used to purify phosphoglycerate kinase from a variety of sources. The choice of buffer pH for the chromatography was made according to the relative electrophoretic mobility of the enzyme from the species concerned. 2. Outlines of the methods used to isolate the enzyme from over 20 sources are presented. The enzyme was purified from the muscle tissue of a variety of mammals, fish and birds, from liver of several animals, from yeast, Escherichia coli, and plant leaves. The more acidic varieties of the enzymes were purified by conventional gradient elution from ion-exchangers as affinity elution procedures were not applicable. 3. The structural and kinetic parameters investigated show that phosphoglycerate kinase is evolutionarily a highly conservative enzyme; there were few differences in properties regardless of source or function (glycolytic, gluconeogenic or photosynthetic). 4. A detailed comparison of the enzyme preparations purified from bovine muscle and bovine liver failed to detect any significant differences between them; the evidence indicates that they are genetically identical.
A new intermediate of the aldolase reaction, the pyruvaldehyde-aldolase-orthophosphate complex
Authors: Grazi E , Trombetta G
Published: August 2015

Fructose 1,6-bisphosphate aldolase from rabbit muscle forms by reaction with dihydroxyacetone phosphate a pyruvaldehyde-aldolase-orthophosphate complex that is in equilibrium with the eneamine intermediate. The new intermediate accumulates in two phases. The first one is practically complete in 40ms, and the second occurs with an apparent first-order rate constant of 4.6 +/- 0.5s-1. The new intermediate breaks down slowly with the release into the medium of pyruvaldehyde and Pi. The rate of the spontaneous release is higher at acidic than at neutral pH.
Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart
Authors: Malkinson A M , Gharrett A J , Hogy L
Published: August 2015

1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase ‘microheterogeneity”, defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific.
Topography of the rhodopsin molecule. Identification of the domain phosphorylated
Authors: Sale G J , Towner P , Akhtar M
Published: August 2015

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.
Purification and properties of nitrite reductase from Escherichia coli K12
Authors: Coleman K J , Cornish-Bowden A , Cole J A
Published: August 2015

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2′-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.
Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa
Authors: Philippe M , Chevaillier P
Published: August 2015

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.
The assembly of early components of complement on antibody-antigen aggregates and on antibody-coated erythrocytes
Authors: Goers J W F , Porter R R
Published: August 2015

Radioimmune assays were developed to assay the binding of complement components C1q, C1s and C4 to antibody aggregates and to cell-bound antibody. The binding of the components was compared with the haemolytic activity and with the capacity to form the C3 convertase activity in the presence of excess C2. The destruction of whole complement and of C4 activity is similar per 1,000 molecules of antibody in aggregates and cell-bound antibody, as is the binding of C1g and C1s, the latter being in a 1:2 molar ratio. The binding of C4 is about 12 times greater, per 1,000 molecules of antibody, on cells than in aggregates. However, the effective C4 molecules, as judged by the formation of C3 convertase activity, are much more similar on cells and aggregates. An assembly mechanism of the early components of complement on antibody-coated cells, which is compatible with these results, is suggested.