bluuebunny/crossref_metadata_2025 huggingface dataset:
Alcoholysis of the endogenous phosphate esters in rats treated with large doses of ethanol
Authors: Tomaszewski M. , Buchowicz J.
Published: August 2015

1. Monoethyl phosphate was isolated from the liver of rats treated with large doses of ethanol. The14C- and32P-labelled products were obtained when [2-14C]ethanol and [32P]orthophosphate respectively were used as the radioactive precursors. 2. The isolated ethyl phosphate preparations were identified by their chemical properties, chromatographic behaviour and enzymic hydrolysis, which, for the14C-labelled substrate, resulted in a partial recovery of the administered [14C]ethanol. 3. The possibility of artifact formation of ethyl phosphate was excluded by suitable control experiments. 4. It is concluded that ethyl phosphate formed in vivo may be a product of phosphate-catalysed alcoholysis of various phosphate esters. The physiological significance of the possible substitution of water by ethanol in reactions catalysed by hydrolytic enzymes under conditions of acute body intoxication with the alcohol is emphasized.
Active-site labelling of triose phosphate isomerase. The reaction of bromohydroxyacetone phosphate with a unique glutamic acid residue and the migration of the label to tyrosine
Authors: De La Mare Susan , Coulson A. F. W. , Knowles J. R. , Priddle J. D. , Offord R. E.
Published: August 2015

Triose phosphate isomerase from chicken muscle reacts stoicheiometrically with the active-site-directed irreversible inhibitor bromohydroxyacetone phosphate with concomitant loss of all catalytic activity. The primary site of attachment has been shown to be a unique glutamic acid residue in the sequence Ala-Tyr-Glu-Pro-Val-Trp. Unless the inhibitor–enzyme bond is stabilized by reduction of the C-2 carbonyl group with borohydride, the phosphate group is lost and the label migrates to the adjacent tyrosine residue. It is suggested that the γ-carboxylate group of the glutamic acid residue may be the base responsible for primary proton abstraction from substrate in the catalysis. The failure of this reagent specifically to inactivate either muscle or yeast aldolase, and the use of the reagent in preparing isomerase-free glycolytic enzymes, is discussed.
Effect of hexachlorobenzene on haem synthesis
Authors: Rajamanickam C. , Amrutavalli J. , Rao M. R. S. , Padmanaban G.
Published: August 2015

Several drugs are known to induce the liver microsomal mixed-function oxidase system when administered in vivo or even in vitro in cell culture. A sequence of events has been suggested in which the drug is visualized to induce δ-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, which is followed by enhanced haem synthesis and cytochrome P-450 content, facilitating the increase in the drug-metabolizing activity of the liver microsomal fraction. The present studies show that the fungicide hexachlorobenzene, when administered to female rats, can lead to enhanced amounts and rate of synthesis of cytochrome P-450 under conditions when the rate of total haem synthesis has not appreciably altered. The subsequent increase in the rate of total haem synthesis as well as the initial increase in amounts of cytochrome P-450 are brought about under conditions when δ-aminolaevulinate synthetase activity remains constant. However, manifestation of porphyria due to prolonged drug administration is accompanied by a twofold increase in δ-aminolaevulinate synthetase activity. The increase in enzyme activity appears to be due to a decreased degradation rate of the enzyme.
The relation of sodium and potassium ion transport to the respiration and adenine nucleotide content of liver slices treated with inhibitors of respiration
Authors: Van Rossum G. D. V.
Published: August 2015

1. The dependence of the net transport of Na+and K+by rat liver on the respiration has been determined by incubating slices in the presence of varying concentrations of respiratory inhibitors. 2. Neither the rate of net transport nor the total amount of each ion transported was inhibited unless the rate of endogenous respiration was decreased below a critical value of about 330mmol of O2/h per kg of protein (i.e. 50% of the total endogenous respiration). 3. The uninhibited rate of respiration could be varied over a twofold range (380–770mmol of O2/h per kg of protein) by the use of different substrates, but the critical value for the onset of transport inhibition was quite constant (290–360mmol/h per kg of protein) under these different conditions. 4. Slices incubated at 38°C without inhibitors showed an increase of their ATP content and the concentration ratio ATP/ADP. The final ATP content and concentration ratio, ATP/ADP, of slices treated with different concentrations of inhibitors were closely related to the rate of respiration. 5. The increased ATP content of the control slices during incubation was equal to the increase of total adenine nucleotides. At increasing degrees of respiratory inhibition the relative contributions of ADP and AMP to the total adenine nucleotide content increased. 6. The critical rate of respiration for the onset of inhibition of ion transport and the corresponding contents of adenine nucleotides provide estimates of the maximal values of certain parameters of energy metabolism required for the support of alkali-cation transport in the liver slices.
Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores
Authors: Guillory Richard J. , Fisher Ronald R.
Published: August 2015

Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing Pi as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg2+concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores.
S-Adenosylmethionine decarboxylase from human prostate. Activation by putrescine
Authors: Zappia Vincenzo , Cartenì-Farina Maria , Pietra Gennaro Della
Published: August 2015

1. The presence of S-adenosylmethionine decarboxylase in human prostate gland is reported. A satisfactory radiochemical enzymic assay was developed and the enzyme was partially characterized. 2. Putrescine stimulates the reaction rate by up to 6-fold at pH7.5: the apparent activation constant was estimated to be 0.13mm. The stimulation is pH-dependent and a maximal effect is observed at acid pH values. 3. Putrescine activation is rather specific: other polyamines, such as spermidine and spermine, did not show any appreciable effect. 4. The apparent Km for the substrate is 4×10−5m. The calculated S-adenosylmethionine content of human prostate (0.18μmol/g wet wt. of tissue) demonstrates that the cellular amounts of sulphonium compound are saturating with respect to the enzyme. 5. The enzyme is moderately stable at 0°C and is rapidly inactivated at 40°C. The optimum pH is about 7.5, with one-half of the maximal activity occurring at pH6.6. 6. Several carboxy-14C-labelled analogues and derivatives of S-adenosylmethionine were tested as substrates. The enzyme appears to be highly specific: the replacement of the 6′-amino group of the sulphonium compound alone results in a complete loss of activity. 7. Inhibition of the enzyme activity by several carbonyl reagents suggests an involvement of either pyridoxal phosphate or pyruvate in the catalytic process. 8. The inhibitory effect of thiol reagents indicates the presence of ‘essential’ thiol groups.
The effects of amino acids on albumin synthesis by the isolated perfused rat liver
Authors: Kelman L. , Saunders S. J. , Wicht S. , Frith L. , Corrigall A. , Kirsch R. E. , Terblanche J.
Published: August 2015

Albumin synthesis was measured in the isolated perfused rat liver by using the livers of both well-fed and starved rats. Starvation markedly decreased albumin synthesis. The livers from starved rats were unable to increase synthesis rates after the addition to the perfusates of single amino acids or the addition of both glucagon and tryptophan. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine, added together to ten times their normal peripheral blood concentrations, restored synthesis rates to normal. The plasma aminogram (i.e. the relative concentrations, of amino acids) was altered by depriving rats of protein for 48h. The use of blood from the deprived rats as perfusate, instead of normal blood, decreased albumin synthesis rates significantly by livers obtained from well-fed rats. The addition of single amino acids, including the non-metabolizable amino acid, α-aminoisobutyric acid, to the above mixture increased albumin synthesis rates to normal values. It is concluded that amino acids play an important role in the control of albumin synthesis and that more than one mechanism is probably involved.
Purification and properties of l(+)-lactate dehydrogenase from potato tubers
Authors: Davies D. D. , Davies S.
Published: August 2015

1. A purification of l(+)-lactate dehydrogenase is described. 2. The final preparation is active with NADH and NADPH and with a number of keto acids, but evidence is presented to support the view that a single enzyme is involved. 3. NAD+ showed product inhibition, but at slightly acid pH values there was evidence of co-operative binding. 4. At acid pH values ATP was a potent inhibitor and appears to be an allosteric effector. At neutral or alkaline pH values ATP behaved as a weak competitive inhibitor. 5. The physiological significance of inhibition by ATP is discussed.
The fate of cyclamate in man and other species
Authors: Renwick A. G. , Williams R. T.
Published: August 2015

1. 14C-labelled cyclamate has been administered to guinea pigs, rabbits, rats and humans. When given orally to these species on a cyclamate-free diet, cyclamate is excreted unchanged. In guinea pigs some 65% of a single dose is excreted in the urine and 30% in the faeces, the corresponding values for rats being 40 and 50%, for man, 30–50% and 40–60%, and for rabbits, 90 and 5%, the excretion being over a period of 2–3 days. 2. Cyclamate appears to be readily absorbed by rabbits but less readily by guinea pigs, rats and humans. 3. If these animals, including man, are placed on a diet containing cyclamate they develop the ability to convert orally administered cyclamate into cyclohexylamine and consequently into the metabolites of the latter. The extent to which this ability develops is variable, the development occurring more readily in rats than in rabbits or guinea pigs. In three human subjects, one developed the ability quite markedly in 10 days whereas two others did not in 30 days. Removal of the cyclamate from the diet caused a diminution in the ability to convert cyclamate into the amine. 4. In rats that had developed the ability to metabolize orally administered cyclamate, intraperitoneally injected cyclamate was not metabolized and was excreted unchanged in the urine. The biliary excretion of injected cyclamate in rats was very small, i.e. about 0.3% of the dose. 5. The ability of animals to convert cyclamate into cyclohexylamine appears to depend upon a continuous intake of cyclamate and on some factor in the gastrointestinal tract, probably the gut flora.
The metabolism of phenylmercury by the rat
Authors: Daniel J. W. , Gage J. C. , Lefevre P. A.
Published: August 2015

The metabolism of [U-14C]phenylmercury acetate was studied in the rat. After a single subcutaneous dose a small proportion is excreted unchanged in urine, and a larger amount in bile with some resorption from the gut. The greater part of the dose is broken down in the tissues to yield inorganic mercury which is excreted mainly in faeces, and conjugates of phenol and quinol are excreted in urine. In experiments in vitro phenylmercury is broken down by liver homogenates to release inorganic mercury and benzene; this reaction is effected by the soluble, but not the microsomal, fraction and does not require NADPH or NADH. No elemental mercury is formed under these conditions. It is probable that this reaction occurs in vivo and the benzene produced is rapidly converted into phenol and quinol by microsomal enzymes.
Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei
Authors: Austoker J. , Cox D. , Mathias A. P.
Published: August 2015

1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg2+and Mn2+concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of α-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.
Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom
Authors: De Lucca F. L. , Imaizumi M. T.
Published: August 2015

1. The incorporation of [5-3H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (‘milking’) of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10–14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation.
Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings
Authors: Sawhney S. K. , Naik M. S.
Published: August 2015

1. In rice seedlings synthesis of methyl viologen–nitrite reductase was stimulated by light, as was that of NADH–nitrate oxidoreductase (EC 1.6.6.1). A small residual effect of light on the synthesis of the enzymes persisted in the dark for a short time. 2. In etiolated seedlings exposed to light and nitrate, a lag period of 3h was necessary before enzyme synthesis commenced, whereas in green seedlings kept in the dark for 36h, synthesis of both the enzymes started as soon as light and nitrate were provided. 3. Experiments with cycloheximide suggested that fresh protein synthesis in light was necessary for formation of active enzymes. Mere activation by light of inactive enzymes or their precursors, was not involved. 4. In green seedlings synthesis of nitrite reductase was more sensitive to chloramphenicol than that of nitrate reductase. In chloramphenicol-treated etiolated seedlings, however, synthesis of both the enzymes was inhibited to the same extent on subsequent light-treatment. 5. A close correlation was observed between inhibition of the Hill reaction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and simazin [2-chloro-4,6-bis(ethylamino)-s-triazine] (at high concentration) and the inhibition of enzyme synthesis. At lower concentrations, however, simazin stimulated nitrate reductase. 6. In a single leaf synthesis of enzymes was observed only in portions exposed to light, whereas little activity was present in the dark covered part. 7. CO2 deprivation severely inhibited the synthesis of enzymes in the light. Sucrose could not reverse this effect. 8. In excised embryos cultured in synthetic media containing sucrose, light was also essential for enzyme formation. 9. It is suggested that redox changes taking place in the green tissues as a result of the Hill reaction create conditions favourable for the induced synthesis of nitrate reductase and nitrite reductase.
Preparation and some properties of a suspension of fragmented tubules from rat kidney
Authors: Dawson A. G.
Published: August 2015

A method is described whereby short fragments of rat kidney tubule were obtained when kidney slices were gently dispersed by exposure to collagenase and hyaluronidase. When suspended in buffered saline the fragmented tubules respired actively over a period of several hours, the rate of oxygen consumption being proportional to the amount of cell protein. Oxygen uptake was stimulated by the addition of glucose, lactate, butyrate, α-oxoglutarate and other substrates and was decreased by the omission of Ca2+ from the suspending medium. With α-oxoglutarate as the added substrate, dinitrophenol strongly stimulated oxygen uptake. Dinitrophenol had a less-marked stimulatory effect when glucose was the added substrate, and inhibited respiration in the absence of added substrate. Oligomycin inhibited respiration and this inhibition was partially reversed by dinitrophenol. Fragmented tubules synthesized glucose from lactate at a high rate but this capacity for gluconeogenesis was abolished by dinitrophenol and by physically damaging the cells.
The metabolism of fluoroacetate in lettuce
Authors: Ward P. F. V. , Huskisson N. S.
Published: August 2015

1. Whole lettuce plants were incubated with (1) [1-14C]acetate, (2) fluoroacetate followed by [1-14C]acetate, (3) fluoro[1-14C]acetate, (4) fluoro[2-14C]acetate or (5) S-carboxy[14C]methylglutathione. 2. Fluoroacetate did not affect the expiration of 14CO2 from [1-14C]acetate and only a small amount of 14CO2 was produced from either fluoro[1-14C]-acetate or fluoro[2-14C]acetate in 43h. 3. Fluoroacetate at 50mg/kg wet wt. doubled the plant citrate concentration after 43h incubation, and depending on the age and size of the plant 50–100% of the compound was metabolized. 4. With both fluoro[1-14C]acetate and fluoro[2-14C]acetate all the radioactivity except that in the CO2 was found in the water-soluble acid fraction. About 2% was in fluorocitrate and the remainder, apart from unchanged fluoroacetate, was in a number of compounds devoid of fluorine but containing nitrogen and sulphur. These were peptide-like and could be separated by chromatography on an amino acid analyser. 5. Identical compounds were obtained from the spontaneous reaction between iodo[2-14C]acetate and glutathione, the major product being S-carboxymethylglutathione. 6. S-Carboxymethylcysteine was also isolated and its mass spectrum compared with a commercial sample. 7. Reaction rates of all the monohaloacetates with glutathione were studied at pH7 at 25°C. No reaction was observed with fluoroacetate. 8. The metabolism of fluoroacetate by lettuce is discussed in relation to that of aliphatic and aromatic halogen compounds, including fluoroacetate, by mammalian liver and to the metabolism of fluoroacetate by different plants reported by other workers.
Interaction of prostaglandins with blood plasma proteins. Comparative binding of prostaglandins A2, F2α and E2 to human plasma proteins
Authors: Raz Amiram
Published: August 2015

1. The binding of prostaglandin A2 and prostaglandin F2α to human plasma proteins was investigated by DEAE-Sephadex chromatography and polyacrylamide-gel electrophoresis. Both prostaglandins, when added to human plasma in vitro, were found to become bound mainly to plasma albumin. 2. The extent of binding of prostaglandins added to human plasma in low to moderate concentrations was found to be approx. 88, 73 and 58% for prostaglandins A2, E2 and F2α respectively. The order of affinities for the binding of the three prostaglandins to albumin appear to be A2>E2>F2α. 3. The apparent association constants for the binding of these prostaglandins to human serum albumin were estimated to be approx. 4.8×104, 2.4×104 and 0.9×104 litre/mol for prostaglandins A2, E2 and F2α respectively. The results are compared with previously reported association constants for the binding of long-chain fatty acids to both human and bovine albumins.
Ketogenesis from butyrate and acetate by the caecum and the colon of rabbits
Authors: Henning S. J. , Hird F. J. R.
Published: August 2015

1. When studied in vitro, tissue from the caecum and the proximal colon of rabbits converted butyrate into ketone bodies. The conversion was similar to that observed with liver slices. The ketogenic activity was associated with the mucosa rather than the muscle of the gut wall and, in the colon, diminished as the distance from the caecal–colonic junction increased. 2. Tissue from the wall of the ileum, caecum, proximal colon and distal colon was also shown to metabolize [1-14C]butyrate to carbon dioxide. 3. Enzyme assays showed that in both liver tissue and caecal mucosa the activity of hydroxymethylglutaryl-CoA synthase was more than ten times that of acetoacetyl-CoA deacylase. Labelling experiments in vitro gave confirmation of the hydroxymethylglutaryl-CoA pathway. 4. The significance of the conversion of butyrate into ketone bodies is discussed.
Inhibition of cellular transport processes by 5-thio-d-glucopyranose
Authors: Whistler Roy L. , Lake William C.
Published: August 2015

5-Thio-d-glucopyranose, the nearest analogue of normal d-glucose, which is proving a useful tool in examinations of d-glucose biochemistry, affects active and facilitated-diffusion transport processes. 5-Thio-d-glucose is readily transported in rabbit kidney-cortex slices and reaches a tissue/medium ratio of 6.5 within 40min. The sulphur analogue shows typical saturation kinetics with a Km value of 2.4mm and Vmax. value of 70μmol/h per g of cell water. Uptake of 5-thio-d-glucose is phlorrhizin-sensitive, Na+-dependent and energy-dependent. d-Galactose and methyl α-d-glucopyranoside transport is competitively inhibited by 5-thio-d-glucose with Ki values of 4.8 and 9.7mm respectively. 5-Thio-d-glucose thus shows all of the characteristics of active transport in kidney cortex. Transport of neutral amino acids in rat kidney cortex is inhibited by 5-thio-d-glucose. Thus 5.6mm-5-thio-d-glucose causes a 25–30% inhibition of the transport of glycine and the non-metabolized amino acids cycloleucine and α-aminoisobutyric acid. 5-Thio-d-glucose is freely taken up by the facilitated-diffusion transport system in rat diaphragm muscle. The sulphur analogue inhibits the transport of d-xylose in this tissue but has no effect on the uptake of d-arabinose. It is concluded that the ring heteroatom is not an effector of binding in the transport processes examined and causes no important alteration in the conformation of the sugar. The diabetogenic action produced by 5-thio-d-glucose is due, in part, to the ability of the analogue to interfere with cellular transport processes that use d-glucose.
Choline metabolism in the cerebral cortex of guinea pigs. Phosphorylcholine and lipid choline
Authors: Dowdall M. J. , Barker L. A. , Whittaker V. P.
Published: August 2015

1. The labelling of phosphorylcholine and choline-containing phospholipids in the subcellular fractions of guinea-pig cerebral cortex after the intraventricular injection of [N-Me-3H]choline into conscious animals has been studied. Special emphasis was placed upon the synaptosome fraction and early time-periods after administration. 2. The labelling of phosphorylcholine was rapid compared with that of phospholipid and was confined to two distinct subcellular fractions: the soluble cytoplasmic fraction and the synaptosome fraction. Most of the labelled phosphorylcholine of the synaptosome fraction was readily released by osmotic rupture indicating location in the nerve-ending cytoplasm. The two pools of phosphorylcholine had similar specific radioactivities at all observed times. 3. 3H-labelled phospholipid was found in all membranous fractions. The labelling was confined to choline-containing phospholipids, notably phosphatidylcholine. 4. The labelling of the different membranous fractions was similar. 5. The half-life of the choline-containing phospholipids in the synaptic vesicle fraction was very much greater than the acetylcholine in this fraction. 6. Evidence is presented that synthesis de novo of phosphatidylcholine at nerve terminals occurs in vivo.
Studies on the heterogeneity of subfragment-1 preparations. Isolation of a new proteolytic fragment of the heavy chain of myosin
Authors: Stone D. , Perry S. V.
Published: August 2015

1. The physical, chemical and enzymic properties of subfragment 1 prepared from myosin of rabbit skeletal muscle by using two different concentrations of insoluble papain were compared. 2. Subfragment 1 prepared by using a myosin/papain ratio of 2000: 1 (by wt.) migrated on electrophoresis in non-dissociating conditions as a single enzymically active band. When prepared with a myosin/papain ratio of 200: 1 the preparation consisted of two enzymically active components of slightly different electrophoretic mobility. 3. The two types of preparation were obtained in similar yield and possessed similar specific adenosine triphosphatase activities when determined in the presence of Ca2+. 4. Gel electrophoresis in the presence of 8m-urea showed that both preparations contained three light components. The component of molecular weight 15500 was apparently identical with one of the light-chain components of myosin (Ml1). The other two light-chain components of subfragment 1 were not identical with any of the light-chain components of myosin. 5. The heavy-chain fraction of subfragment 1 prepared by using low concentrations of papain dissociated into components with molecular weights of 87000, 69000 and 26000 on electrophoresis in sodium dodecyl sulphate. The heavy-chain fraction of subfragment 1 prepared by using higher concentrations of papain contained components with molecular weights of 69000 and 53000 and relatively increased amounts of the component of molecular weight 26000. 6. The isolated 26000 dalton component had an amino acid composition similar to that of the heavy-chain fraction of subfragment 1 and contained 3-methylhistidine and mono-and tri-Nε-methyl-lysine. It was homogeneous on electrophoresis in the presence of sodium dodecyl sulphate but gave two bands on electrophoresis in 8m-urea.
Cathepsin A in human brain and spleen (Short Communication)
Authors: Bowen D. M. , Davison A. N.
Published: August 2015

A striking loss of cathepsin A activity occurs when relatively crude preparations of the enzyme are maintained at even a mildly basic pH value. This may explain the failure of others to detect the enzyme.
Nucleotide sequence analysis with polynucleotide kinase and nucleotide ‘mapping’ methods. 5′-Terminal sequence of deoxyribonucleic acid from bacteriophages λ and 424
Authors: Murray Kenneth
Published: August 2015

The polynucleotide kinase reaction was used in analyses of complex mixtures of oligodeoxynucleotides which were fractionated by various two-dimensional nucleotide ‘mapping’ procedures. Parallel ionophoretic analyses on DEAE-cellulose paper, pH2, and AE-cellulose paper, pH3.5, of venom phosphodiesterase partial digests of 5′-terminally labelled oligonucleotides enabled the sequence of the nucleotides to be deduced uniquely. A ‘diagonal ionophoresis’ method has been used with mixtures of nucleotides. Application of these methods to 5′-terminally labelled DNA from bacteriophage λ gave the terminal sequences pA-G-G-T-C-G and pG-G-G-C-G. Identical 5′-terminal sequences were found with DNA from bacteriophage 424.
Polynucleotides associated with histone preparations
Authors: Greenaway Peter J. , Murray Kenneth
Published: August 2015

Histone fractions prepared from various sources by acid extraction or by salt dissociation contained small quantities of ribo- and deoxyribo-nucleotides. These nucleotides were small and heterogeneous and were efficiently removed from histone preparations by ionexchange chromatography. They probably represent contaminants, rather than uniquely complexed nucleotides, of all the histone preparations.
Equilibrium binding of nicotinamide nucleotides to lactate dehydrogenases
Authors: Stinson Robert A. , Holbrook J. John
Published: August 2015

1. No discontinuities were observed during the continuous titration with NADH of the lactate dehydrogenases of ox muscle, pig heart, pig muscle, rabbit muscle, dogfish muscle or lobster tail muscle. The binding was monitored by either the enhanced fluorescence of bound NADH or the quenched fluorescence of the protein. A single macroscopic dissociation constant, independent of protein concentration, could be used to describe the binding to each enzyme, and there was no need to postulate the involvement of molecular relaxation effects. 2. The affinity for NADH decreases only threefold between pH6 and 8.5. Above pH9 the affinity decreases more rapidly with increasing pH and is consistent with a group of about pK9.5 facilitating binding. Muscle enzymes bind NADH more weakly than does the pig heart enzyme. 3. Increasing temperature and increasing concentrations of ethanol both weaken NADH binding. 4. NADH binding is weakened by increasing ionic strength. NaCl is more effective than similar ionic strengths derived from sodium phosphate or sodium pyrophosphate. 5. Commercial NAD+ quenches the protein fluorescence of the heart and muscle isoenzymes. Highly purified NAD+ does not, and its binding was monitored by competition for the NADH-binding sites. A single macroscopic dissociation constant is sufficient to describe NAD+ binding at the concentrations tested. The dissociation constant is about 0.3mm and is not sensitive to changed ionic strength and to changed pH in the range pH6–8.5.
The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase
Authors: Holbrook J. John , Stinson Robert A.
Published: August 2015

1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3μm and from the muscle isoenzyme 25μm at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme–NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme–NADH–substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD+. The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.
Activities of enzymes responsible for steroid biosynthesis and cholesterol ester metabolism in rabbit ovarian interstitial tissue and corpora lutea. A comparison of enzyme activities with flow rates
Authors: Flint A. P. F. , Armstrong D. T.
Published: August 2015

A method involving the use of isolated cholesterol ester-storage granules as substrate is described for the assay of cholesterol esterase in rabbit ovarian tissues. Activities of cholesterol esterase 100–200-fold higher than those previously reported in ovarian tissues were measured by using this method. In addition to that of cholesterol esterase, activities of cholesterol ester synthetase, cholesterol side-chain cleavage enzyme and 3β-hydroxy steroid dehydrogenase were determined in rabbit ovarian interstitial tissue and corpora lutea. Activities of these enzymes are in general compatible with the flows through them measured under a variety of conditions both in vivo and in vitro. It is concluded that, in the rabbit ovarian tissues investigated, these enzymes are capable of catalysing the conversions usually attributed to them.
Hepatic lipid droplets. Isolation, morphology and composition
Authors: DiAugustine Richard P. , Schaefer Joan-Marie , Fouts James R.
Published: August 2015

The floating lipid layer isolated centrifugation of rat liver was examined for composition and ultrastructure. It was chiefly composed of triglycerides and cholesterol esters plus much smaller amounts of free cholesterol, diglycerides, phospholipid and protein. No free fatty acids were detected. The triglyceride and cholesterol ester fractions consisted mostly of esters of linoleic acid, oleic acid and palmitic acid. Electron micrographs of the floating lipid layer revealed numerous spherical osmiophilic droplets having a mean diameter of 0.5–2μm with a very-thin dense outer coat. Similar structures were observed as organelles in electron micrographs of the intact liver cell. The amount of triglyceride in the layer decreased in rats starved for 72h, but pellet triglyceride (homogenate minus the floating lipid layer) was unchanged. These results suggest that the floating lipid layer is the representative in vitro of lipid-rich organelles which probably function as a depot form of hepatic-cell neutral lipid.
Adipose-tissue lipoprotein lipase activity and cellularity in the genetically obese Zucker rat (fa/fa) (Short Communication)
Authors: de Gasquet P. , Péquignot E. , Lemonnier D. , Alexiu A.
Published: August 2015

The lipoprotein lipase activity per adipocyte was increased in the genetically obese rat (fa/fa). However, there was no difference between obese and lean animals when the enzyme activities were related to adipocyte surface area. The possible implications of the findings are discussed.
Phosphorylation of high-molecular-weight membrane protein species in Chinese-hamster ovary cells in culture: effect of 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate plus testosterone (Short Communication)
Authors: Rieber Manuel , Bacalao Josefina
Published: August 2015

Growth of Chinese-hamster ovary cells in [32P]phosphate and [3H]leucine and subsequent assay of the plasma membranes reveals phosphorylation in two protein regions corresponding to molecular weights of 280000 and 195000. Culture in the presence of the 6-N,2-O′-dibutyryl derivative of cyclic AMP plus testosterone does not stimulate [32P]-phosphate incorporation, but determines a modification in the qualitative pattern of phosphorylation.
Control of ornithine decarboxylase activity in stimulated human lymphocytes by putrescine and spermidine
Authors: Kay John E. , Lindsay Valerie J.
Published: August 2015

Ornithine decarboxylase activity in extracts of phytohaemagglutinin-stimulated human lymphocytes is rapidly and extensively inhibited by additions of micromolar concentrations of putrescine or spermidine to the culture medium. This inhibition is not due to feedback inhibition of the enzyme by putrescine, spermidine or their metabolites. Inhibition is dependent on the continuation of protein synthesis, but does not require RNA synthesis. The effect of putrescine is abolished when its conversion into spermidine by the cells is prevented.
Food particles as a site for biohydrogenation of unsaturated fatty acids in the rumen (Short Communication)
Authors: Harfoot Christopher G. , Noble Raymond C. , Moore John H.
Published: August 2015

On incubation of linoleic acid with strained rumen contents from sheep, it was observed that conversion of linoleic acid into C18:1trans-11 monoenoic acid and subsequently into stearic acid was largely associated with the food-particle fraction. The bacteria, protozoa and cell-free supernatant together contributed less than 30% to the overall change in the added C18:2 fatty acid.
Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols
Authors: Brocklehurst Keith , Little Graham
Published: August 2015

1. The u.v.-spectral characteristics of 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2), 2,2′-dipyridyl disulphide (2-Py–S–S–2-Py), 4,4′-dipyridyl disulphide (4-Py–S–S–4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py–2-SH) and 4-thiopyridone (Py–4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5–8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py–2-SH and Py–4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs2 is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5–8 at 25°C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py–S–S–2-Py and 4-Py–S–S–4-Py in the pH range 2.5–8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine–2-Py–S–S–2-Py reaction (kpH4/kpH8=0.015) and for the papain–4-Py–S–S–4-Py reaction (kpH4/kpH8=0.06) are less than 1, that for the papain–2-Py–S–S–2-Py reaction is greater than 1 (kpH4/kpH8=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py–S–S–2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.
The purification and properties of a single chicken pepsinogen fraction and the pepsin derived from it
Authors: Green Margaret L. , Llewellin Joanna M.
Published: August 2015

1. Evidence is given for the presence of at least five pepsinogens in a crude extract of mixed chicken stomachs. One of these was purified and could be activated to yield a single pepsin. 2. The molecular weights of the pepsinogen and pepsin were 36000 and 34000 respectively. The pepsin associated at low pH values and low ionic strength. 3. The amino acid analyses of both proteins are given. The pepsin was devoid of phosphate but contained carbohydrate. 4. The N-terminal amino acids of pepsinogen and pepsin were serine and threonine respectively. Five amino acids were released by carboxypeptidase A and it was deduced that serine may be the C-terminal one. 5. Each protein contained one thiol group per molecule as determined by titration with p-chloromercuribenzoate. The rate of the reaction was very rapid with pepsin, but much slower with pepsinogen, although the same group appeared to react in both instances. The enzymic activity of pepsin was unaffected by the modification. 6. The isoionic point of the pepsin was close to pH4.0 and the enzyme was stable for long periods at pH values up to 7.0. 7. The enzyme hydrolysed bisphenyl sulphite almost as rapidly as did pig pepsin A.
Observations on the action of limit dextrinases on amylopectin-like polysaccharides
Authors: Dunn G. , Hardie D. G. , Manners D. J.
Published: August 2015

The rate of hydrolysis of amylopectin by three different limit dextrinase preparations is only about 15–23% of that of amylopectin β-limit dextrin under similar conditions. On dilution of the enzymes there was no change in specificity. The factors controlling the specificity of the enzyme and the possible significance in vivo of the results are discussed.
Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats
Authors: Gonzalez-Mujica F. , Mathias A. P.
Published: August 2015

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.
The synthesis of esters of bilirubin
Authors: Hutchinson David W. , Johnson Brian , Knell Alan J.
Published: August 2015

1-Alkyl-3-p-tolytriazenes were used to synthesize the methyl, ethyl, isopropyl and benzyl esters of bilirubin. Treatment of a chloroform solution of bilirubin with the triazene at room temperature gave high yields of the corresponding esters. These were identified by n.m.r. and mass spectroscopy together with elemental analysis. N.m.r. studies also suggest that bilirubin dimethyl ester is in the lactam rather than the lactim form.
Some parameters relevant to affinity chromatography on immobilized nucleotides
Authors: Lowe C. R. , Harvey M. J. , Craven D. B. , Dean P. D. G.
Published: August 2015

1. The suitability of cellulose and Sepharose as supports for affinity chromatography of two groups of cofactor-linked enzymes, dehydrogenases and kinases, was examined. Sepharose was found to be superior. 2. The selective capacities of the columns were measured by frontal analysis and are discussed in relation to the nucleotide contents. 3. The effect of various concentrations of enzyme and of non-specific protein on the performance of the affinity columns, and the effects of equilibration time, flow rate, sample volume and dilution of the nucleotide were examined. 4. The effect of interposing polymethylene and polyglycine extension arms between the matrix backbone and the nucleotide was investigated for several cofactor-dependent enzymes. Maximum binding was observed with an extension arm 0.8–1nm long.
Covalent chromatography. Preparation of fully active papain from dried papaya latex
Authors: Brocklehurst Keith , Carlsson Jan , Kierstan Marek P. J. , Crook Eric M.
Published: August 2015

1. A Sepharose–(glutathione–2-pyridyl disulphide) conjugate has been prepared. 2. Its use in a new type of chromatography, covalent chromatography by thiol–disulphide interchange, is described. 3. With this technique, papain containing 1 intact catalytic site [thiol with high reactivity towards 2,2′-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4] per mol of protein is readily prepared both from dried papaya latex and from commercial 2×crystallized partially active papain. 4. The catalysis of the hydrolysis of α-N-benzoyl-l-arginine ethyl ester at pH6.0, 25.0°C, I=0.3 by fully active papain thus prepared is characterized by Km=18.2±<0.1mm and kcat.=16.4±0.5s-1.