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Showing papers in "Biochemical Journal in 1967"


Journal ArticleDOI
TL;DR: Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation, and the resolution is greater and more detailed than by centrifugations, and many samples can be analysed simultaneously and rapidly.
Abstract: 1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.

1,956 citations


Journal ArticleDOI
TL;DR: The bearing of these findings on various problems, including the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of theredox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate.
Abstract: 1. The concentrations of the oxidized and reduced substrates of the lactate-, β-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD+ and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bucher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD+]/[NADH] ratio in the cytoplasm, the β-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38°; I0·25). 3. The mean [NAD+]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7·0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD+]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that β-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD+]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD+]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD+ is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD+–NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.

1,671 citations


Journal ArticleDOI
TL;DR: The acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine, and the method was applied for the determination of Cysteine in perchloric acid extracts of rat brain, liver and blood.
Abstract: 1. An acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine. 2. The method was highly sensitive for the determination of cysteine (in 28.0x10(3)). Homocysteine, glutathione, proline, ornithine and other naturally occurring amino acids tested did not give a similar reaction. 3. The reaction product was stable for at least 3-4hr. at room temperature and the extinction was proportional to the concentration in the range 0.05-0.5mumole of cysteine. 4. The acid ninhydrin reagent also gave yellow products (E(max.) 370-404mmu) with tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and indol-3-ylacetic acid. 5. The method was applied for the determination of cysteine in perchloric acid extracts of rat brain, liver and blood.

1,104 citations


Journal ArticleDOI
TL;DR: Stoicheiometric acid-base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.
Abstract: 1 Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H+ (or OH−) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes 2 The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and β-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively These quotients are constant between pH5·5 and 8·5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation 3 Apparent changes in the →H+/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of →H+/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations 4 Apparent changes in the →H+/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na+ These apparent changes of →H+/O are due to an increase in the rate of the pH-driven decay of the acid pulses 5 The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial →H+/O quotients The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial →H+/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism 6 Stoicheiometric acid–base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3

418 citations


Journal ArticleDOI
TL;DR: Highly purified chromaffin granules can be obtained from homogenates of either ox, pig, horse or rat adrenal medullae by ultracentrifugation of the large-granule fraction layered on 1.6m-sucrose solution, by using angle-head rotors.
Abstract: Highly purified chromaffin granules can be obtained from homogenates of either ox, pig, horse or rat adrenal medullae by ultracentrifugation of the large-granule fraction layered on 1·6m-sucrose solution, by using angle-head rotors. The chromaffin granules are obtained as a pink sediment that is only slightly contaminated by mitochondria and lysosomes.

405 citations


Journal ArticleDOI
TL;DR: Pulsed acid-base titrations of suspensions of rat-liver mitochondria under anaerobic equilibrium conditions show fast and slow titration processes that will account quantitatively for the observed respiratory control in state 4, assuming that oxidoreduction and phosphorylation are coupled by a circulating proton current as required by the chemi-osmotic hypothesis.
Abstract: 1. Pulsed acid-base titrations of suspensions of rat-liver mitochondria under anaerobic equilibrium conditions show fast and slow titration processes. 2. The fast process is the titration of the outer aqueous phase of the mitochondria, which is continuous with the suspension medium, and the slow process can be identified with the titration of the inner aqueous phase of the mitochondria, which is separated from the outer aqueous phase by the non-aqueous osmotic barrier or M phase of the cristae membrane system. 3. The buffering power of the outer and inner phases have been separately measured over a range of pH values. 4. The rate of titration of the inner aqueous phase under a known protonmotive force across the M phase has been characterized by an effective proton conductance coefficient, which, near pH7 and at 25 degrees , is only 0.45mumho/cm.(2) of the M-phase membrane. 5. The low effective proton conductance of the M phase will account quantitatively for the observed respiratory control in state 4, assuming that oxidoreduction and phosphorylation are coupled by a circulating proton current as required by the chemi-osmotic hypothesis. 6. The addition of 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) at normal uncoupling concentrations causes a large increase in the effective proton conductance of the M phase of the cristae membrane. 7. The increase of the effective proton conductance of the M phase by 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) will account quantitatively for the short-circuiting effect of the uncoupling agent on the proton current and for the observed rise of the rate of respiration to that characteristic of state 3 or higher.

380 citations


Journal ArticleDOI
TL;DR: It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage.
Abstract: 1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [(14)C]bicarbonate. 2. Pyruvate-carboxylase activity in both foetal and adult liver occurs mainly in mitochondrial and nuclear fractions, with about 10% of the activity in the cytoplasm. 3. Similar studies of the intracellular distribution of phosphoenolpyruvate carboxykinase show that more than 90% of the activity is in the cytoplasm. However, in the 17-day foetal liver about 90% of the activity is in mitochondria and nuclei. 4. Pyruvate-carboxylase activity in both particulate and soluble fractions is very low in the 17-day foetal liver and increases to near adult levels before birth. 5. Phosphoenolpyruvate-carboxykinase activity in the soluble cell fraction increases 25-fold in the first 2 days after birth. This same enzyme in the mitochondria has considerable activity in the foetal and adult liver and is lower in the newborn. 6. Kinetic and other studies on the properties of phosphoenolpyruvate carboxykinase have shown no differences between the soluble and mitochondrial enzymes. 7. It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage.

368 citations


Journal ArticleDOI
TL;DR: Two sites of action of glucagon must therefore be postulated: one concerned with mobilization of liver glycogen, the other with the promotion of gluconeogenesis.
Abstract: 1. The rates of gluconeogenesis from many precursors have been measured in the perfused rat liver and, for comparison, in rat liver slices. All livers were from rats starved for 48hr. Under optimum conditions the rates in perfused liver were three to five times those found under optimum conditions in slices. 2. Rapid gluconeogenesis (rates of above 0.5mumole/g./min.) were found with lactate, pyruvate, alanine, serine, proline, fructose, dihydroxyacetone, sorbitol, xylitol. Unexpectedly other amino acids, notably glutamate and aspartate, and the intermediates of the tricarboxylic acid cycle (with the exception of oxaloacetate), reacted very slowly and were not readily removed from the perfusion medium, presumably because of permeability barriers which prevent the passage of highly charged negative ions. Glutamine and asparagine formed glucose more readily than the corresponding amino acids. 3. Glucagon increased the rate of gluconeogenesis from lactate and pyruvate but not from any other precursor tested. This occurred when the liver was virtually completely depleted of glycogen. Two sites of action of glucagon must therefore be postulated: one concerned with mobilization of liver glycogen, the other with the promotion of gluconeogenesis. Sliced liver did not respond to glucagon. 4. Pyruvate and oxaloacetate formed substantial quantities of lactate on perfusion, which indicates that the reducing power provided in the cytoplasm was in excess of the needs of gluconeogenesis. 5. Values for the content of intermediary metabolites of gluconeogenesis in the perfused liver are reported. The values for most intermediates rose on addition of lactate. 6. The rates of gluconeogenesis from lactate and pyruvate were not affected by wide variations of the lactate/pyruvate ratio in the perfusion medium.

354 citations


Journal ArticleDOI
TL;DR: It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.
Abstract: 1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the α-glycerophosphate–dihydroxyacetone phosphate and malate–oxaloacetate cycles in this tissue.

354 citations


Journal ArticleDOI
TL;DR: The purified protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume, which was compatible with the protein's having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.
Abstract: 1. A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25% of the total protein of the granules and gave a single band on gel electrophoresis. 2. The protein is unusually rich in acidic amino acids, notably glutamic acid (26·0%, w/w); it is also relatively rich in proline (8·6%, w/w) but poor in cystine (0·35%, w/w). 3. A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. 4. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. 5. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume. 6. The hydrodynamic properties of the protein, but not its molecular weight, were dependent on the ionic strength of the solvent. Increasing the ionic strength caused an increase in the sedimentation and diffusion coefficients, but a decrease in the intrinsic viscosity and in the frictional ratio of the protein. 7. Optical-rotatory-dispersion measurements indicated that only a small part of the polypeptide chain was in an α-helical conformation. 8. These results are compatible with the protein's having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.

313 citations


Journal ArticleDOI
TL;DR: The results suggest that acetoacetate can supply energy for the basal requirements and for gluconeogenesis but not for the secretory work, and indicate that, in the rat, the kidney is a major source of body glutamine.
Abstract: 1 A technique for perfusing the isolated rat kidney is described It is primarily designed for the study of renal metabolism but is also suitable for studying some aspects of the secretory function; this was normal with respect to minimal glucosuria The glomerular filtration rate as measured by creatinine clearance was lower than in vivo and slowly decreased with time 2 Gluconeogenesis from a variety of precursors was rapid and similar to that in kidney-cortex slices, in contrast with liver where the perfused organ is more effective than slices Whereas the maximal rates of gluconeogenesis from glycerol and pyruvate were similar in liver and kidney, the rates from succinate, malate and fumarate were 14–20 times, and those from glutamate and aspartate about three times, as high in the kidney 3 The oxygen consumption of the perfused organ was about twice that of cortex slices, presumably because of the secretory work done in the perfused organ but not in slices 4 The rate of acetoacetate oxidation was about the same in the perfused organ and in slices but, because of the higher rate of oxygen consumption, the percentage contribution of acetoacetate to the fuel of respiration was lower in the perfused organ The results suggest that acetoacetate can supply energy for the basal requirements and for gluconeogenesis but not for the secretory work 5 Glutamine was formed at a high rate from glutamate and at a lower rate from aspartate The high rates indicate that, in the rat, the kidney is a major source of body glutamine

Journal ArticleDOI
TL;DR: Rat-liver supernatant catalyses the reaction of a number of other alphabeta-unsaturated compounds, including aldehydes, ketones, lactones, nitriles and nitro compounds, with glutathione: separate enzymes may be responsible for these reactions.
Abstract: 1. Rat-liver supernatant catalyses the reaction of diethyl maleate with glutathione. 2. Evidence is presented that the enzyme involved is different from the known glutathione-conjugating enzymes, glutathione S-alkyltransferase, S-aryltransferase and S-epoxidetransferase. 3. Rat-liver supernatant catalyses the reaction of a number of other alphabeta-unsaturated compounds, including aldehydes, ketones, lactones, nitriles and nitro compounds, with glutathione: separate enzymes may be responsible for these reactions.

Journal ArticleDOI
TL;DR: The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultraneous residue was confirmed, confirming the trend for stepwise successive increase or decrease in the relative amounts of the main constituents of lipoproteins.
Abstract: 1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.

Journal ArticleDOI
TL;DR: In both liver and adipose tissue from ruminants, acetate is a more important source of lipid than glucose, and two enzymes of the hexose monophosphate shunt, glucose 6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogensase, are active in both tissues and from the three species.
Abstract: 1. The incorporation of labelled glucose into lipid by liver slices from sheep and cows is considerably less than that by liver slices from the rat, although oxidation to carbon dioxide occurs to a similar extent. ATP citrate lyase and NADP malate dehydrogenase are inactive in both sheep and cow liver but active in rat liver. The absence of the citrate-cleavage pathway of lipogenesis in ruminant liver has been confirmed by the negligible amounts of C-3 of aspartate incorporated into fatty acids. 2. Considerable amounts of [14C]acetate are incorporated into fatty acids and non-saponifiable lipid in rat and ruminant liver. Acetyl-CoA synthetase, the initial enzyme in the metabolism of acetate, has a high activity in liver from rat and ruminants. 3. In adipose tissue from ruminants more acetate than glucose is converted into lipids, whereas the converse is true in rat adipose tissue. The greater incorporation of [14C]acetate into fatty acids in adipose tissue from the ruminant as compared with the non-ruminant may be caused, in part, by the higher activity of acetyl-CoA synthetase activity in the ruminant. 4. The results suggest that, in both liver and adipose tissue from ruminants, acetate is a more important source of lipid than glucose. 5. Two enzymes of the hexose monophosphate shunt, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, are active in both tissues and from the three species.

Journal ArticleDOI
TL;DR: Comparison of the acetylation states of carnitine and CoA in perfused hearts suggests that the carn itine acetyltransferase reactants may remain near equilibrium despite wide variations in their steady-state concentrations.
Abstract: 1. Free carnitine, acetylcarnitine, short-chain acylcarnitine and acid-insoluble carnitine (probably long-chain acylcarnitine) have been measured in rat tissues. 2. Starvation caused an increase in the proportion of carnitine that was acetylated in liver and kidney; at least in liver fat-feeding had the same effect, whereas a carbohydrate diet caused a very low acetylcarnitine content. 3. In heart, on the other hand, starvation did not cause an increase in the acetylcarnitine/carnitine ratio, whereas fat-feeding caused a decrease. The acetylcarnitine content of heart was diminished by alloxan-diabetes or a fatty diet, but not by re-feeding with carbohydrate. 4. Under conditions of increased fatty acid supply the acid-insoluble carnitine content was increased in heart, liver and kidney. 5. The acylation state of carnitine was capable of very rapid change. Concentrations of carnitine derivatives varied with different methods of obtaining tissue samples, and very little acid-insoluble carnitine was found in tissues of rats anaesthetized with Nembutal. In liver the acetylcarnitine (and acetyl-CoA) content decreased if freezing of tissue samples was delayed; in heart this caused an increase in acetylcarnitine. 6. Incubation of diaphragms with acetate or dl-β-hydroxybutyrate caused the acetylcarnitine content to become elevated. 7. Perfusion of hearts with fatty acids containing an even number of carbon atoms, dl-β-hydroxybutyrate or pyruvate resulted in increased contents of acetylcarnitine and acetyl-CoA. Accumulation of these acetyl compounds was prevented by the additional presence of propionate or pentanoate in the perfusion medium; this prevention was not due to extensive propionylation of CoA or carnitine. 8. Perfusion of hearts with palmitate caused a severalfold increase in the content of acid-insoluble carnitine; this increase did not occur when propionate was also present. 9. Comparison of the acetylation states of carnitine and CoA in perfused hearts suggests that the carnitine acetyltransferase reactants may remain near equilibrium despite wide variations in their steady-state concentrations. This is not the case with the citrate synthase reaction. It is suggested that the carnitine acetyltransferase system buffers the tissue content of acetyl-CoA against rapid changes.


Journal ArticleDOI
TL;DR: A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye-histone complexes formed in the gel, and the relative mobilities with respect to a marker protein, bovine plasma albumin.
Abstract: 1. A new method has been devised for the separation of the histone fractions of calf thymus by electrophoresis in polyacrylamide gel at pH2·4. 2. The fractions have been characterized by their relative mobilities with respect to a marker protein, bovine plasma albumin. 3. A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye–histone complexes formed in the gel.

Journal ArticleDOI
TL;DR: Water-soluble alcohols can markedly stimulate the liberation of choline from ultrasonically treated lecithin by phospholipase D, usually due to an increase in hydrolase activity although often the associated transferase activity contributes.
Abstract: 1. Purified phospholipase D can catalyse the transfer of a ;phosphatidyl' unit from lecithin to various aliphatic alcohols such as glycerol, ethanolamine, methanol and ethylene glycol with the formation of the equivalent phospholipid. 2. The transferase reaction occurs simultaneously with hydrolase activity but at high alcohol concentrations the former predominates. 3. The acceptor molecule must contain a primary alcoholic grouping. 4. The chromatographic and ionophoretic mobilities of the deacylation products of many enzymically synthesized phospholipids are reported. 5. Enzymically prepared phosphatidylglycerol has been isolated in good yield. Chemical degradation showed that the ;phosphatidyl unit' of lecithin had been transferred predominantly to the alpha-hydroxyl groups of glycerol. 6. Water-soluble alcohols can markedly stimulate the liberation of choline from ultrasonically treated lecithin by phospholipase D. The stimulation is usually due to an increase in hydrolase activity although often the associated transferase activity contributes.

Journal ArticleDOI
TL;DR: A labelling pattern typical of sugar-cane was found in several species of Gramineae but not in others, and of 16 species from other Families only a species of Cyperaceae contained a large proportion of the fixed radioactivity in oxaloacetate, malate and aspartate.
Abstract: 1. The pathway of photosynthesis in sugar-cane, which gives most of the radio-activity fixed during short periods in (14)CO(2) in C-4 of oxaloacetate, malate and aspartate, was examined under varied conditions. 2. The pattern of labelling was essentially the same with leaves of different ages and with leaves equilibrated at carbon dioxide concentrations in the range 0-3.8% (v/v) and light-intensities in the range 1400-9000ft.-candles before adding (14)CO(2). 3. Radioactive products were examined after exposing leaves of 33 different plant species to (14)CO(2) for 4sec. under standard conditions. 4. A labelling pattern typical of sugar-cane was found in several species of Gramineae but not in others. Of 16 species from other Families only a species of Cyperaceae contained a large proportion of the fixed radioactivity in oxaloacetate, malate and aspartate.

Journal ArticleDOI
TL;DR: Phosphopyruvate carboxylase was apparently the major photosynthetic carbon dioxide-fixing enzyme in the tropical grasses, although malic enzyme may contribute to a lesser extent.
Abstract: 1. The activity per unit of chlorophyll of certain carboxylases, and of other enzymes involved in photosynthesis, was determined in leaf extracts of the tropical grasses, sugar-cane, maize and sorghum, and compared with the activities for wheat, oat and silver-beet. Maximum rates of photosynthetic carbon dioxide uptake were also measured for comparison with enzyme activities. 2. Phosphopyruvate carboxylase activity was about 60 times greater in the tropical grasses than in wheat, oat and silver-beet and was severalfold higher than the rates of photosynthetic carbon dioxide uptake. Most of the enzyme was located in the chloroplast fraction of cell extracts. 3. Phosphopyruvate carboxylase was apparently the major photosynthetic carbon dioxide-fixing enzyme in the tropical grasses, although malic enzyme may contribute to a lesser extent. 4. Tropical grasses contained less than one-tenth of the ribulose diphosphate carboxylase activity present in wheat, oat and silver-beet. For the tropical grasses this activity, determined with a saturating concentration of bicarbonate, was approx. 10% of the rate of photosynthesis. 5. The fraction-1 protein content of leaf extracts paralleled the ribulose diphosphate carboxylase activity. 6. In contrast, the activity of several other enzymes of the Calvin cycle was similar in the different species examined.

Journal ArticleDOI
TL;DR: Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest.
Abstract: 1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7·6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.

Journal ArticleDOI
TL;DR: The separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous FIColl gradient, and the neuronal fraction was richer than the glial in all except phospholipid.
Abstract: 1. A procedure has been developed for the separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex. Separation depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous Ficoll gradient. Up to 1·5×107 neuronal cells could be collected from 12 brains within 3hr. The morphological appearance of these cells seemed good, and the fraction was 8·5-fold purified in terms of dry weight. Average dry weight per neuron was 2300μμg. Maximum glial contamination of the neuronal fraction was 11% as determined by carbonic anhydrase measurements. The glial fraction was free from neurons but contained various subcellular contaminants. 2. Concentrations of nucleic acids, phospholipid, protein and phosphoprotein were determined in the separated fractions. The neuronal fraction was richer than the glial in all except phospholipid. Succinate dehydrogenase was equally distributed between neurons and glia but the neuronal fraction was 1·8-fold enriched in cytochrome oxidase. 3. Measurement of respiration by the cells showed an endogenous uptake of 117mμmoles of oxygen/mg./hr. in neurons, and 173mμmoles of oxygen/mg./hr. in glia. Addition of substrate at 10mm stimulated uptake to similar values in both fractions. With glucose it was 390, with pyruvate 355, and with glutamate 215mμmoles of oxygen/mg./hr. This represented a larger stimulation of neuronal than of glial respiration compared with the basal level. 4. Respiration in cell suspensions was 70–80% of that of slices, whereas fractionated tissue homogenates had respiratory rates of only one-third those of the cell suspensions. Lactate dehydrogenase content of cell suspensions was maintained during gradient centrifugation and washing. 5. The possible uses of isolated cell preparations are discussed.

Journal ArticleDOI
TL;DR: It is suggested that mobilization of free fatty acids in diabetes leads to the synthesis of additional glyceride in muscle, which could account for the balance of oxygen consumption in the normal or diabetic heart perfused with glucose and insulin.
Abstract: 1. Methods are described for the extraction of lipid and assay of mono-, di- and tri-glyceride glycerol and phospholipid phosphorus in rat heart and gastrocnemius muscles. 2. In hearts from normal animals, concentrations found were: monoglyceride, 0·6; diglyceride, 0·1; triglyceride, 12·6μmoles of glyceride glycerol/g. of dry muscle; phospholipid, 171μg.atoms of phospholipid phosphorus/g. of dry muscle. Concentrations of glycerides in gastrocnemius muscle were similar to heart muscle but those of phospholipids were lower (64μg.atoms of phospholipid phosphorus/g. of dry muscle). 3. Alloxan-diabetes increased the concentration of triglyceride in the muscles twofold. This increase was shown to be dependent in the heart on the availability of growth hormone and cortisol but not on the availability of dietary lipid. Total glyceride in the heart was increased after 48 and 72hr. starvation but not after 96hr. Changes in glyceride concentration seen in starvation and diabetes were not associated with significant changes in phospholipid concentration. It is suggested that mobilization of free fatty acids in diabetes leads to the synthesis of additional glyceride in muscle. 4. The possible contribution of glyceride fatty acid in the heart to respiration during perfusion has been calculated from the net loss of glyceride during perfusion, and also from the relative rates of lipolysis and esterification and compared with oxidation of fatty acid required for the balance of oxygen consumption (oxygen not utilized in the oxidation of glucose or glycogen glucose). In the normal or diabetic heart perfused with glucose and insulin the breakdown of glyceride can account for the balance of oxygen consumption. In the normal heart perfused without substrate the balance of oxygen consumption is not entirely accounted for by the breakdown of glyceride.

Journal ArticleDOI
TL;DR: Changes in the activity of hepatic citrate-cleavage enzyme (ATP-citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis, however, NADP-malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.
Abstract: 1. Lipogenesis, as measured by the incorporation of (14)C-labelled glucose or acetate into fatty acids in liver slices, is high in foetal and adult rat liver but is low in the liver of the suckling rat, especially with glucose as substrate. 2. The rate of synthesis of non-saponifiable lipids from glucose is about 15 times as great in the liver of the 18-day foetus as in adult liver. Activity in the newborn is negligible. 3. Glucose incorporation into fat is strongly concentration-dependent in liver slices from the adult and 2-week-old rat, but less markedly so in liver slices from the foetus. 4. Changes in the activity of hepatic citrate-cleavage enzyme (ATP-citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis. However, NADP-malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.

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TL;DR: In this article, the authors measured milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-(14)C]stearate, oleate and palmitate into fed lactating goats.
Abstract: 1. Measurements were made of milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-(14)C]stearate, [U-(14)C]oleate, [U-(14)C]palmitate and [1-(14)C]acetate into fed lactating goats. 2. Entry rates of fatty acids into the circulation were 4.2mg./min./kg. body wt. for acetate, and 0.18, 0.28 and 0.42mg./min./kg. for stearate, oleate and palmitate respectively. Acetate accounted for 23% of the total carbon dioxide produced by the whole animal, and contributed to the oxidative metabolism of the mammary gland to about the same extent. Corresponding values for each of the long-chain acids were less than 1%. 3. There were no significant arteriovenous differences of phospholipids, sterols or sterol esters, and their fatty acid composition showed no net changes during passage through the mammary gland. 4. There were large arteriovenous differences of plasma triglycerides, and their fatty acid composition showed marked changes across the gland. The proportions of palmitate and stearate fell, and that of oleate increased. 5. Arteriovenous differences of plasma free fatty acids (FFA) were small and variable, but a large fall in the specific radioactivity of each of the long-chain acids examined indicated substantial uptake of plasma FFA, accompanied by roughly equivalent FFA release from mammary tissue. The uptake of FFA was confirmed by the extensive transfer of radioactivity into milk. The FFA of milk were similar in composition and radioactivity to the milk triglyceride fatty acids, and quite unlike plasma FFA. 6. The formation of large amounts of oleic acid (18-21 mg./min.) from stearic acid was demonstrated. 7. During the terminal stages of the [(14)C]acetate infusion, milk triglyceride fatty acids of chain length C(4)-C(14) showed specific radioactivities that were 75-90% of that of blood acetate, and that of palmitate was roughly one-quarter of this value. Oleate and stearate were unlabelled. 8. The results confirmed that milk fatty acids of chain length C(4)-C(14) arise largely from blood acetate, and palmitate is derived partly from acetate and partly from plasma triglyceride, the latter fraction being almost the sole precursor of oleate and stearate.

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TL;DR: Evidence is presented demonstrating that part or all of the prostaglandsin A2 is formed during the isolation procedures from endogenous prostaglandin E2.
Abstract: Rabbit kidney medulla (10kg) was homogenized in 5mm-disodium hydrogen phosphate and deproteinized with ethanol, and the concentrated supernatant solution was extracted at pH8 with light petroleum and at pH2 with chloroform The acidic lipids present in the chloroform phase were separated on silicic acid columns into three biologically active fractions The first fraction contained only vasodepressor activity; the second fraction contained both vasodepressor and non-vascular-smooth-muscle-stimulating activity; the third fraction contained both vasopressor and non-vascular-smooth-muscle-stimulating activity Purification of each fraction by reversed-phase partition and thick-layer chromatography yielded three pure acids Thin-layer chromatographic, spectroscopic and mass-spectral analysis of the acids and their methyl esters established their structures as prostaglandins E2, F2α and A2 Evidence is presented demonstrating that part or all of the prostaglandin A2 is formed during the isolation procedures from endogenous prostaglandin E2

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TL;DR: In rat epididymis, there is no significant beta-glucosid enzyme activity, nor is there appreciable inhibition of the beta-galactosidase and beta-d-fucosidsase activities of the preparation by gluconolactone.
Abstract: 1. In barley, beta-glucosidase and beta-galactosidase are separate enzymes. The former also displays beta-d-fucosidase activity. 2. In the limpet, Patella vulgata, beta-glucosidase activity is associated with the beta-d-fucosidase, previously shown to be a separate entity from the beta-galactosidase also present. 3. Almond emulsin presents all three activities as a single enzyme. Each is equally inhibited by glucono-, galactono- and d-fucono-lactone. 4. In rat epididymis, there is no significant beta-glucosidase activity, nor is there appreciable inhibition of the beta-galactosidase and beta-d-fucosidase activities of the preparation by gluconolactone.

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TL;DR: The results are consistent with the view that alkaline phosphatases are also inorganic pyrophosphatases, and the two types of activity were not separated by gel filtration or by anion-ex exchange or cation-exchange chromatography.
Abstract: 1. The inorganic-pyrophosphatase activity of alkaline phosphatases prepared from human liver and small intestine was investigated at different stages of purification. 2. Both liver and intestinal preparations possessed pyrophosphatase activity at all stages of purification, and the two types of activity were not separated by gel filtration or by anion-exchange or cation-exchange chromatography. 3. After starch-gel electrophoresis of the tissue extracts, the zones of pyrophosphatase activity coincided exactly with alkaline-phosphatase zones. 4. Hydrolysis of each type of substrate was inhibited by the presence of the other, and a constant ratio of alkaline-phosphatase activity to pyrophosphatase activity was maintained during inactivation of the enzymes by incubation at 55°. 5. These results are consistent with the view that alkaline phosphatases are also inorganic pyrophosphatases.

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TL;DR: DNA has been isolated from different mammalian tissues and the DNA preparations were free from RNA, protein and polysaccharides and have a similar range of sedimentation coefficients.
Abstract: 1. DNA has been isolated from different mammalian tissues. The DNA preparations were free from RNA, protein and polysaccharides and have a similar range of sedimentation coefficients (approx. 24s). 2. Protein was removed by a two-stage extraction with a phenol-cresol mixture by using a detergent with 4-aminosalicylate in the first stage and sodium chloride in the second. 3. Polysaccharides remained in solution when DNA was precipitated with 2-butoxyethanol in the presence of 0.5m-sodium chloride and 1.5m-sodium benzoate. 4. Ribosomal RNA was removed by precipitation in the presence of 3m-sodium chloride at 0 degrees , when DNA remained soluble.

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TL;DR: The use of 1,3,5-tri-(p-oc-L-fucosyloxyphenylazo)2,4,6-trihydroxybenzene for precipitation of an L- fucose-binding protein from the seed of Lotu8 tetragonolobu8 revealed that some polysaccharide components present in aqueous extracts of various seeds are precipitated with this dye and with the related glucoside dye.
Abstract: The use of 1,3,5-tri-(p-oc-L-fucosyloxyphenylazo)2,4,6-trihydroxybenzene for precipitation of an L-fucose-binding protein from the seed of Lotu8 tetragonolobu8 (Yariv, Kalb & Katchalski, 1967) revealed that some polysaccharide components present in aqueous extracts of various seeds are precipitated with this dye and with the related glucoside dye 1,3,5-tri-(p-fl-D-glucosyloxyphenylazo) 2,4,6 trihydroxybenzene (Yariv, Rapport & Graf, 1962). We observed that arabic acid is also precipitated with the glucoside dye. In this communication we describe the precipitation reaction and a procedure for the isolation of some seed polysaccharides based on this reaction. Precipitation of arabic acid (Pfanstiehl Laboratories Inc., Waukegan, Ill., U.S.A.) by the glucoside dye was effected at room temperature in 0-1Msodium phosphate buffer, pH6-8, in the range of 5-100 ,g. of polysaccharide/ml. A critical ratio of dye to arabic acid is necessary for precipitation. A plot ofprecipitated dye versus added dye is concave upwards at low dye concentration and becomes concave downwards at higher concentration, eventually reaching an asymptote. The asymptotic value of precipitated dye is proportional to the amount of polysaccharide. In an experiment, glucoside dye was added to each ofa series ofconical test tubes containing 40,ug. of arabic acid in 2ml. After 1 hr. the mixtures were centrifuged and the concentration of dye in the supernatants was measured. Precipitates were observed in all mixtures containing more than 12,ug. of added dye. The saturation value of 41 ,g. of bound dye was reached with 100,ug. of added dye and persisted up to 400,ug. of added dye. With a 1: 1 weight ratio of dye to polysaccharide more than 90% of the polysaccharide was found in the precipitate by means of a procedure described below for the isolation of the dye-binding polysaccharides. The dye did not precipitate arabic acid in 10% (w/v) NaCl or in 8M-urea. Heating an aqueous solution of arabic acid in a boiling-water bath for 1 hr. destroyed its ability to be precipitated with the dye. The following seed materials were used for isolation of the dye-binding polysaccharides: soyabean flour was a defatted product marketed as Soyafluff 200W by Central Soya, Chicago, Ill.,