bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of potato lectin and the nature of its glycoprotein linkages
Authors: Allen Anthony K. , Desai Nila N. , Neuberger Albert , Creeth J. Michael
Published: August 2015

1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer–dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000–34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the β-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although α-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of β-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single α-galactopyranoside residues that can be removed by the action of α-galactosidase from coffee beans but not by a β-galactosidase. This is the first report of an α-galactoside linkage to serine. The effect of α-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of β-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues had been removed. This inhibitory effect of the arabinofuranoside residues on the β-elimination reaction is likely to be due to a negative charge on the hydroxy groups of the adjacent arabinofuranoside residues, which would be ionized at this high pH value. 6. It is suggested that potato lectin may be representative of a class of soluble plant glycoproteins that would include precursors of the cell-wall glycoprotein extensin. If this is the case, extensin should also contain β-l-arabinofuranosides linked to hydroxyproline and α-d-galactopyranosides linked to serine residues of the polypeptide chain.
Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver
Authors: Parker P J , Randle P J
Published: August 2015

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.
Effect of parathyrin on the transport properties of isolated renal brush-border vesicles
Authors: Evers C , Murer H , Kinne R
Published: August 2015

The transport properties of brush-border membrane vesicles isolated by a calcium-precipitation method from the renal cortex of normal and parathyrin (parathyroid hormone)-treated rats were studied by a rapid-filtration technique. Parathyrin elicited a dose-dependent decrease in the Na+-dependent phosphate uptake by the brush-border membrane vesicles, but the uptake of D-glucose, Na+ and mannitol was not affected. A maximum inhibition of 30% was observed after the application of 30 U.S.P. units intramuscularly 1 h before the animals were killed. Intravenous infusion of dibutyryl cyclic AMP (0.5-1.5 MG) also decreased the phosphate uptake by the brush-border vesicles. Both dibutyryl cyclic AMP and parathyrin were ineffective when added in vitro to brush-border membrane vesicles isolated from normal rats. These data suggest that parathyrin exerts its action on the phosphate reabsorption in the renal proximal tubule by affecting the Na+/phosphate co-transport system in the brush-border membrane. The effects of parathyrin on Na+ and glucose transport, however, seem to be due to alterations to the driving forces for transport and not to the brush-border transport systems.
Studies on the orientation of brush-border membrane vesicles
Authors: Haase W , Schäfer A , Murer H , Kinne R
Published: August 2015

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.
Possible interrelationship between vitamins E and b12 in the disturbance in methylmalonate metabolism in vitamin E deficiency
Authors: Pappu A S , Fatterpaker P , Sreenivasan A
Published: August 2015

1. The disturbance in 2-methylmalonate metabolism resulting in its increased urinary excretion observed in vitamin E deficiency is not caused by increased formation of methylmalonate from propionate as is evident from the activity of the enzyme propionyl-CoA carboxylase (EC 6.4.1.3), but can be traced to an impairment in the conversion of methylmalonate into succinate by the vitamin B12-requiring enzyme, methylmalonyl-CoA mutase (EC 5.4.99.2) in rat liver. 2. It is shown that the decrease in the activity of methylmalonyl-CoA mutase in vitamin E deficiency is not a consequence of a secondary vitamin B12 deficiency. Peroxidative destruction of the coenzyme in vitamin E deficiency was also ruled out. The results suggest a defect in the conversion of cyanocobalamin into its coenzyme form.
The effect of dietary protein deficiency on albumin synthesis and on the concentration of active albumin messenger ribonucleic acid in rat liver
Authors: Pain V M , Clemens M J , Garlick P J
Published: August 2015

In rats fed on a protein-deficient diet, albumin synthesis as a percentage of total liver protein synthesis falls from the normal value of approx. 15% to about 8%. We have extracted total cytoplasmic RNA from individual rat livers and measured the concentration of active albumin mRNA by translation in a reticulocyte lysate system from which the endogenous mRNA had been removed [Pelham & Jackson (1976) Eur. J. Biochem. 67, 247-256]. In this messenger-dependent system it is possible to measure the synthesis of albumin as a proportion of the overall protein synthesis promoted by the addition of the hepatic RNA. The results show that the concentration of translatable albumin mRNA in samples of total cytoplasmic RNA from livers of protein-deficient rats is decreased markedly. These findings suggest that dietary protein supply affects selectively the synthesis and/or functional stability of albumin mRNA in rat liver.
The temperature-dependence of the loss of latency of lysosomal enzymes
Authors: Ruth Robin C. , Weglicki William B.
Published: August 2015

1. When Triton-filled lysosomes from rat liver are incubated for up to 50min at 37°C, pH7.4, in 0.25m-sucrose, no loss of latency of N-acetyl-β-glucosaminidase or p-nitrophenyl phosphatase occurs unless the incubated lysosomes are cooled to approx. 15°C. 2. It is suggested that a phase change takes place in the incubated lysosomal membranes on cooling; it starts at approx. 15°C and probably is not complete at 0°C. 3. Incubation of the lysosomes causes an increased potential for loss of latency of the lysosomal enzymes. This potential is not fully expressed at elevated temperature (e.g. 37°C), but is expressed on cooling. 4. The increase at elevated temperature in potential for loss of latency exhibits biphasic kinetics, with an initial rapid phase followed by a slower phase, which is linear with respect to time. The extra loss of latency resulting from the rapid phase in proportional to the temperature of the incubation. 5. Arrhenius plots of the increase is potential for loss of latency during the slow phase for N-acetyl-β-glucosaminidase and p-nitrophenyl phosphatase exhibit marked deviations from linearity beginning at approx. 15°C. This suggests that the increase in potential for loss of latency is affected by a phase change that occurs around this temperature. 6. Activation energies for the increase in potential for loss of latency at and above 22°C are 53.1±5.4kJ/mol (12.7±1.3kcal/mol) for N-acetyl-β-glucosaminidase and 45.2±7.5kJ/mol (10.8±1.8kcal/mol) for p-nitrophenyl phosphatase. It is postulated that these energies reflect enzymic action, the products of which cause loss of latency to occur on cooling.
The properties of adenosine triphosphatase from exponential and synchronous cultures of Alcaligenes eutrophus H16
Authors: Edwards Clive , Spode Juliet A. , Jones Colin W.
Published: August 2015

The properties of Alcaligenes eutrophus ATPase (adenosine triphosphatase) were investigated by using subcellular fractions prepared from cells growing in exponential and synchronous cultures. Both the soluble and membrane-bound forms of the ATPase were inhibited non-competitively (Ki 142μm) by Nbf-Cl (4-chloro-7-nitrobenzofurazan), whereas only the membrane-bound enzyme was inhibited (non-competitive; Ki 750μm) by NN′-dicyclohexylcarbodi-imide. Neither the activity of the ATPase nor its sensitivity to these two inhibitors varied during exponential growth. However, marked variations in ATPase activity were observed during synchronous growth, which were characterized by maxima at approx. 0.4 and 0.9 of a cell cycle and minima at approx. 0.1 and 0.6 of a cycle. Sensitivity to Nbf-Cl and NN′-dicyclohexylcarbodi-imide also varied during the cell cycle; maximum inhibition by the former occurred at approx. 0.4 and 0.9 of a cell cycle, whereas maximum inhibition by the latter was located at approx. 0.1 and 0.6 of a cell cycle. Proton conductance by whole cells was also periodic during the cell cycle, the lowest rates occurring at approx. 0.15 and 0.55 of a cycle and the highest rates at approx. 0.4 and 0.9 of a cycle, but →H+/O quotients for the oxidation of endogenous substrates remained relatively constant and indicated the presence of four proton-translocating respiratory segments throughout the cell cycle. These results are discussed in terms of ATPase and respiratory-chain structure and function during the cell cycle of Alcaligenes eutrophus.
The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen
Authors: Vaes G , Eeckhout Y , Lenaers-Claeys G , François-Gillet C , Druetz J E
Published: August 2015

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.
Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier
Authors: Halestrap A P
Published: August 2015

The effects of exchangeable ions and pH on the efflux of pyruvate from preloaded mitochondria are reported. Efflux obeys first-order kinetics, and the stimulation of efflux by exchangeable ions such as acetoacetate and lactate obeys Michaelis–Menten kinetics. The apparent Km value +/- S.E. for acetoacetate was 0.56 +/- 0.14 mM (n = 5) and that for lactate 12.3 +/- 2.3 mM (n = 6). The Vmax. values +/- S.E. at 0 degrees C were 16.2 +/- 2.0 and 21.9 +/- 2.7 nmol/min per mg of protein. The exchange of a variety of other substituted monocarboxylates was also studied. Efflux was also stimulated by increasing the external pH. The data gave a pK for the transport process of 8.35 and a Vmax. of 3.31 +/- 0.14 nmol/min per mg. The similarity of the Vmax. values for various exchangeable ions but the difference of this from the Vmax. in the absence of exchangeable ions may indicate that transport of pyruvate occurs with H+ and not in exchange for an OH- ion. The inhibition of transport by alpha-cyano-4-hydroxycinnamate took several seconds to reach completion at 0 degrees C. It is proposed that inhibition occurs by binding to the substrate site and subsequent reaction with an -SH group on the inside of the membrane. The inhibitor can be displaced by substrates that can also enter the mitochondria independently of the carrier and so compete with the inhibitor for the substrate-binding site on the inside of the membrane. A mechanism for transport is proposed that invokes a transition state of pyruvate involving addition of an -SH group to the 2-carbon of pyruvate. Evidence is presented that suggests that ketone bodies may cross the mitochondrial membrane either on the carrier or by free diffusion. The physiological involvement of the carrier in ketone-body metabolism is discussed. The role of ketone bodies and pH in the physiological regulation of pyruvate transport is considered.
Liver enzyme induction by 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) is accompanied by an increase in the specific activity of elongation factor 1
Authors: Young E T , Nicholls D M
Published: August 2015

Homogenates of liver were obtained from control rats and from rats that had received DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. The postmicrosomal supernatant fractions were used for the purification of elongation factor 1 by hydroxyapatite chromatography and phosphocellulose chromarography. The amount of binding factor present was essentially the same for both groups of animals, but the specific activity, as measured by the binding assay, was about twice as high in the DDT-treated preparations. After sucrose-gradient sedimentation, the difference in specific activity was found to reside in the low-molecular-weight (50000) form of elongation factor 1. The implications of an increased reactivity of elongation factor 1 during the induction of membrane enzymes are discussed.
Increased calcium-ion influx is a component of capacitation of spermatozoa
Authors: Singh J P , Babcock D F , Lardy H A
Published: August 2015

Capacitation (modifications required for gamete fusion) is produced by incubating guinea-pig spermatozoa in vitro in a chemically defined medium. It is shown that during such incubation a net uptake of Ca2+ by the sperm occurs in two distinguishable phases. An initial loose association of Ca2+, possibly to surface sites, is unaffected by agents (Mg2+, inhibitors of mitochondiral function) that prevent or delay the exocytotic spermatozoal acrosome reaction. The time course of a secondary Ca2+ uptake parallels or slightly precedes the time course of the acrosome reaction. This parallelism is maintained during a variety of treatments that either expedite (local anaesthetics, ionophore A23187, Triton X-100) or delay (Mg2+, low external Ca2+) the acrosome reaction. We conclude that the secondary Ca2+ influx described herein apparently serves to link alterations of the spermatozoal membrane to subsequent contractile and secretory components of the capacitation sequence.
Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium
Authors: Hemmilä I A , Mäntsälä P I
Published: August 2015

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.
Primary structures of cysteine-containing peptides from the calcium ion-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum
Authors: Allen G , Green N M
Published: August 2015

A preliminary investigation of the primary structure of the Ca(2+-transporting ATPase (adenosine triphosphatase) protein of rabbit skeletal-muscle sarcoplasmic reticulum is reported. The preparation of derivatives of delipidated protein in a form suitable for sequence analysis is described. Tryptic peptides containing S-carboxymethylcysteine residues were isolated from the reduced carboxymethylated protein, and their sequences were partially determined. The results are consistent with mol.wt. about 105000 for the polypeptide, and the absence of extended repeated lengths of sequence. The distribution of tryptophan and cysteine residues between large, aggregated peptides and soluble tryptic peptides shows that these residues are concentrated in different regions of the primary structure. This observation agrees with other evidence that these residues are, on the whole, widely separated in the native protein. The details of the procedures used to isolate the peptides, and the evidence for the determination of their sequences, are given Supplementary Publication SUP 50085 (30 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem.J. (1978) 169, 5.
The amino acid sequence of ferredoxin from the red alga Porphyra umbilicalis
Authors: Takruri I , Haslett B G , Boulter D , Andrew P W , Rogers L J
Published: August 2015

The amino acid sequence of the ferrodoxin of Porphyra umbilicalis was determined by the dansyl-phenyl isothiocyanate method, on peptides obtained by tryptic, chymotryptic and thermolytic digestion of the protein or its CNBr-cleavage fragments. The molecule consists of 98 residues, has an unblocked N-terminus and shows considerable similarity with other plant-type ferredoxins. It is the first reported sequence of a red-algal ferredoxin.
Comparative study of the glycosphingolipids of chicken bursa of Fabricius and of chicken, rat and human thymus
Authors: Narasimhan R , Murray R K
Published: August 2015

1. The glycosphingolipid compositions of the thymus and bursa of Fabricius of young male chickens were compared. The two tissues were found to contain complex mixtures of both neutral glycosphingolipids and gangliosides. Both tissues contained mono-, di-, tri-, tetra- and penta-glycosylceramides; the pentaglycosylceramide displayed a reaction of identity with authentic Forssman antigen when tested against a specific anti-(Forssman antigen) serum. The ganglioside G(m3) containing N-acetylneuraminic acid was the principle ganglioside of both tissues. 2. The thymus contained appreciable amounts of the simple ganglioside N-acetylneuraminylgalactosylceramide, a compound not found in the bursa. The ganglioside G(d3) (disialohaematoside) was detected in both tissues. 3. Rat and human thymus, like sheep thymus (Narasimhan, Hay, Greaves & Murray (1976) Biochim, Biophys. Acta 431, 578-591), both contained a tetraglycosylceramide species as their most complex neutral glycosphingolipid and possessed little or no Forssman antigen. They also contained a complex mixture of gangliosides. 4. The possible significance of these results is briefly discussed.
Studies on the tissue specificity of the high-mobility-group non-histone chromosomal proteins from calf
Authors: Rabbani A , Goodwin G H , Johns E W
Published: August 2015

The high-mobility-group (HMG) non-histone chromosomal proteins from calf thymus, liver, spleen and kidney were extracted, and fractionated by CM-Sephadex chromatography and trichloroacetic acid precipitation. The isolated proteins HMG 1, HMG 2 and HMG 17 from the tissues were compared by polyacrylamide-gel electrophoresis, isoelectric focusing and amino acid analysis. The results show that the three proteins are very similar in the tissues studied, implying a lack of tissue specificity.
The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout
Authors: Skinner E R , Rogie A
Published: August 2015

1. VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins were isolated from the serum of trout (Salmo gairdneri Richardson). 2. Each lipoprotein class resembled that of the human in immunological reactivity, electrophoretic behaviour and appearance in the electron microscope. Trout LD lipoprotein, however, was of greater density than human LD lipoprotein. 3. The trout lipoproteins have lipid compositions which are similar to those of the corresponding human components, except for their high contents of long-chain unsaturated fatty acids. 4. HD and LD lipoproteins were immunologically non-identical, whereas LD lipoproteins possessed antigenic determinants in common with VLD lipoproteins. 5. VLD and HD lipoproteins each contained at least seven different apoproteins, whereas LD liprotein was composed largely of a single apoprotein which resembled human apolipoprotein B. 6. At least one, and possibly three, apoprotein of trout HD lipoprotein showed features which resemble human apoprotein A-1.7. The broad similarity between the trout and human lipoprotein systems suggests that both arose from common ancestral genes early in evolutionary history.
Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat
Authors: Hutson N J , Kerbey A L , Randle P J , Sugden P H
Published: August 2015

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.
The direct determination of porphyrin carboxylic acids. High-pressure liquid chromatography with solvent systems containing phase-transfer agents
Authors: Bonnett R , Charalambides A A , Jones K , Magnus I A , Ridge R J
Published: August 2015

A novel method for separating porphyrin polycarboxylic acids is described and illustrated by its application to the direct analysis of biological (deep-sea medusae), clinical (urine) and chemical (‘haematoporphyrin derivative’) samples.
Solubilization of bovine corpus-luteum adenylate cyclase in lubrol-PX, triton X-100 or digitonin and the stabilizing effect of sodium fluoride present in the solubilization medium
Authors: Young J L , Stansfield D A
Published: August 2015

1. Adenylate cyclase activity of the washed 600g sediment of bovine corpus-luteum homogenate was solubilized by Lubrol-PX, Triton X-100 and digitonin. Digitonin was the least destructive of NaF-stimulated activity. 2. NaF, present in the solubilization medium together with MgSO4, increased the percentage yields of soluble activity from untreated 600g sediment and 600g sediment which had been preincubated with p[NH]ppG (guanosine 5′-[betagamma-imido]triphosphate). The stabilizing influence of NaF was most marked with digitonin. However, the highest specific activities of soluble enzyme were obtained with Lubrol-PX as solubilizing agent, since digitonin solubilized more membrane protein than does Lubrol-PX, and less of the activity of the digitonin-dispersed 600g sediment was recovered in the 105000g supernatant. 3. p[NH]ppG also has a stabilizing effect when present during the solubilization, but less so than NaF. 4. Both NaF and MgSO4 alone have a stabilizing effect during solubilization. The greatest amounts of soluble activity were obtained with both agents present in the solubilization medium, there being a synergistic effect.
Reversal by Triton WR-1339 of ethynyloestradiol-induced hepatic cholesterol esterification
Authors: Davis Roger A. , Showalter Radene , Kern Fred
Published: August 2015

Rats treated with ethynyloestradiol have marked hypolipidaemia: serum cholesterol is decreased to 5%, triacylglycerol to 10% and phospholipid to 70% of control concentrations. Loss of serum cholesterol follows an exponential decay, with a half-life of 1.13±0.09 days. After 4 days of treatment, serum cholesterol concentrations remain relatively constant (ranging from 1 to 20mg/100ml) for at least 30 days. There is a concomitant 20-fold decrease in the d<1.21 fraction of serum proteins and a similar decrease in serum apolipoproteins as measured by sodium dodecyl sulphate/10%-polyacrylamide-gel electrophoresis. The activity of hepatic microsomal acyl-CoA–cholesterol O-acetyltransferase (EC 2.3.1.26) was significantly increased by ethynyloestradiol treatment (P<0.05). This activation caused hepatic cholesteryl esters containing mainly C18:1 fatty acids to increase linearly as serum cholesterol concentrations decreased (r=0.9675, P<0.001). Triton WR-1339, a non-ionic detergent that inhibits lipoprotein catabolism, was used to estimate hepatic lipid secretion by measuring the increment in serum lipids after its administration. At 15h after Triton WR-1339 administration, serum cholesterol concentrations were increased equally in both control and ethynyloestradiol-treated rats. In contrast, the increment of serum triacylglycerol of treated rats was 40% of that found in control rats, indicating that ethynyloestradiol inhibits hepatic triacylglycerol secretion. Triton WR-1339 inhibited the oestrogen activation of hepatic microsomal acyl-CoA–cholesterol O-acyltransferase and restored hepatic cholesteryl ester concentrations to normal values. These data suggest that ethynyloestradiol and its pharmacological ‘antagonist’ Triton WR-1339 alter hepatic triacylglycerol secretion via a mechanism associated with changes in hepatic cholesterol esterification.
Effects of maternal ethanol consumption on hepatic lipid biosynthesis in foetal and neonatal rats
Authors: Rawat A K
Published: August 2015

Effects of prolonged maternal ethanol consumption were studied on hepatic lipid content, on the rates of fatty acid synthesis and on the activities of enzymes involved in fatty acid synthesis in the livers of foetal and suckling neonatal rats. Prolonged maternal ethanol consumption resulted in a significant increase in the contents of hepatic total lipids, triacylglycerols and plasma unesterified fatty acids in foetal and neonatal rats. Studies in vitro with 3H2O showed that maternal ethanol consumption did not result in a significant change in its rate of incorporation into lipid fractions of foetal and neonatal livers. The rates of fatty acid synthesis showed a pronounced decrease immediately after birth, compared with the foetal stage, but increased in the adult animals. On the other hand, the highest rates of lipid oxidation were observed in the neonatal stage. Maternal ethanol consumption resulted in a significant decrease in the rates of [14C]palmitate oxidation to 14CO2 by both the foetal and neonatal livers. Maternal ethanol consumption did not result in an increase in the activities of any of the lipid-synthesizing enzymes tested throughout the period of development. Although increased fatty acid synthesis does not seem to be the mechanism for the accumulation of these lipids, decreased oxidation of the lipids may be partly responsible for the lipid accumulation.
Diurnal variation and response to food withdrawal of lactose synthesis in lactating rats
Authors: Carrick D T , Kuhn N J
Published: August 2015

1. The incorporation of radiolabelled plasma glucose into mammary lactose was used to measure the rate of lactose synthesis in lightly anaesthetized lactating rats. 2. Lactose synthesis showed a diurnal variation with a minimum at 18:00h 3. Food withdrawal for 6h did not affect lactose synthesis in the early morning but greatly decreased it in the afternoon or evening. 4. Plasma glucose, milk sugars and total galactosyltransferase activity (EC 2.4.1.22) did not show the above changes. 5. Measurements of plasma insulin, which varies diurnally, and experiments with injected insulin suggested that variations of insulin within the physiological range do not account for the changes in lactose synthesis described.
Diminished excretion of polyamines from BHK-21/C13 cells exposed to methylglyoxal bis(guanylhydrazone)
Authors: Melvin M A L , Keir H M
Published: August 2015

Methylglyoxal bis(guanylhydrazone) (1,1′-[methylethanediylidine)dinitrilo]diguanidine) inhibited the growth of BHK-21/C13 cells in monolayer cultures. Accumulation of spermidine and spermine was inhibited, whereas the accumulation of putrescine was increased. The intracellular spermidine/spermine molar ratio decreased conly slightly after exposure of the cells to 20 micrometer-methylglyoxal bis(guanylhydrazone) for 1 day. Cells incubated in the presence of the drug released less polyamine into the culture medium that did control cells, the polyamine released consisting almost exclusively of spermidine, both free and as a conjugated form.
Measurement of adenosine 3′:5′-cyclic monophosphate by competitive binding to salt-dissociated protein kinase
Authors: Døskeland S O , Haga H J
Published: August 2015

An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments.
Kinetics of polyamine synthesis and turnover in mouse fibroblasts
Authors: McCormick Frank
Published: August 2015

Kinetics of polyamine synthesis and degradation were studied in mouse fibroblasts growing in suspension culture. The approach was to prelabel cells with radioactive polyamines and to observe the rate of loss of radioactivity and the rate of decrease in specific activity of these compounds in cells. Radioactive putrescine declined with a half-life of 1.5–2h, whether derived directly from exogenous putrescine or indirectly from ornithine. Much of this turnover was due to excretion, the kinetics of which suggested that a steady-state was being established between putrescine inside and outside the cells. Within 5h of medium change, cells growing at a density of 5×105cells/ml had supplied putrescine to the medium to a concentration of about 1μm. When cells were prelabelled with either putrescine or spermidine, radioactivity in cell spermidine declined with a half-life of 60h. This rate of turnover is sufficient to provide all the spermine required by the cell. Spermine synthesis was the only observed reaction of spermidine, although some excretion into the growth medium was detected. Spermine was not degraded at a detectable rate as long as cells were growing exponentially; in stationary phase, degradation to spermidine, which was excreted, became significant. The half-lives of the specific activities of spermine, spermidine and putrescine were 24, 15 and 1.5h respectively. From these values, the rate of synthesis of each was calculated. Spermidine was synthesized at 6.8 times the rate of spermine, and putrescine was synthesized at 0.46nmol/106cells per h, twice the rate of spermidine. The significance of these kinetic parameters is discussed.
Transverse organization of phospholipids across the bilayer of plasma-membrane subfractions of rat hepatocytes
Authors: Higgins J A , Evans W H
Published: August 2015

Phospholipase C treatment of vesicular subfractions of plasma membranes derived from the three functional domains of rat liver indicated that there is an asymmetric distribution of phospholipids across the bilayer of these membranes. The bile-canalicular and sinusoidal membranes were similar and different from the contiguous membrane.
The influence of passive stretch on the growth and protein turnover of the denervated extensor digitorum longus muscle
Authors: Goldspink David F.
Published: August 2015

At 7 days after cutting the sciatic nerve, the extensor digitorum longus muscle was smaller and contained less protein than its innervated control. Correlating with these changes was the finding of elevated rates of protein degradation (measured in vitro) in the denervated tissue. However, at this time, rates of protein synthesis (measured in vitro) and nucleic acid concentrations were also higher in the denervated tissue, changes more usually associated with an active muscle rather than a disused one. These anabolic trends have, at least in part, been explained by the possible greater exposure of the denervated extensor digitorum longus to passive stretch. When immobilized under a maintained influence of stretch the denervated muscle grew to a greater extent. Although this stretch-induced growth appeared to occur predominantly through a stimulation of protein synthesis, it was opposed by smaller increases in degradative rates. Nucleic acids increased at a similar rate to the increase in muscle mass when a continuous influence of stretch was imposed on the denervated tissue. In contrast, immobilization of the denervated extensor digitorum longus in a shortened unstretched state reversed most of the stretch-induced changes; that is, the muscle became even smaller, with protein synthesis decreasing to a greater extent than breakdown after the removal of passive stretch. The present investigation suggests that stretch will promote protein synthesis and hence growth of the extensor digitorum longus even in the absence of an intact nerve supply. However, some factor(s), in addition to passive stretch, must contribute to the anabolic trends in this denervated muscle.
Differential light-scattering of granal and agranal chloroplasts and their fragments
Authors: Faludi-Dániel A , Bialek G E , Horváth G , Sz-Rózsa Z , Gregory R P F
Published: August 2015

Intact (class-A) granal and agranal maize chloroplasts and chloroplast fragments were examined for differential scattering of circularly polarized light (measured at 90 degrees) and c.d. (circular dichroism) (measured at 0 degrees) by using a modified spectropolarimeter with a large acceptance angle. Useful c.d. information was obtained by making corrections for scattered light. Chloroplast fragments exhibited a large and characteristic differential scattering of circularly polarized light recognized in the presence of granal chloroplasts. It is confirmed that agranal chloroplasts do not have the intense 682 nm c.d. peak that is assigned to the presence of grana.
The lipoprotein lipase (clearing-factor lipase) activity of cells isolated from rat cardiac muscle
Authors: Chohan P , Cryer A
Published: August 2015

The total lipoprotein lipase activity recovered in suspension of cells prepared from adult rat hearts was unaffected by the nutritional state of the animals used. The enzyme activity present in the cell suspensions was almost exclusively associated with the cardiac muscle cells present as the major cell type.
The biosynthesis of brain gangliosides. Ganglioside-glycosylating activity in rat brain neuronal perikarya fraction
Authors: Maccioni H J F , Defilpo S S , Landa C A , Caputto R
Published: August 2015

Rat brain homogenate and the synaptosmal and neuronal perikarya fractions from 17-day-old rats were compared for their activities in sialosylating endogenous gangliosides and transferring N-acetylneuraminic acid and galactose to several glycolipids in vitro. The sialosylation of endogenous gangliosides and the activities of sialosyltransferases acting either on lactosylceramide or haematoside as acceptors, as well as galactosyltransferase acting on Tay-Sachs ganglioside as acceptor, were between 3-and 12-fold higher in the neuronal perikarya fraction than in whole homgenate on a protein or ganglioside basis. The activities found in the synaptosomal fraction were negligible. No evidence was found to indicate that the low activities in this fraction were due to the presence of inhibitors of the transfer activities or to inacessibility of the substrates to their respective enzymes. These findings, and the time course of labelling of gangliosides of the neuronal perikarya and synaptosomes from rats that received an injection of N-[3H]acetylmannosamine, indicate that the main cellular site of glycosylation of neuronal gangliosides is in the neuronal perikarya.
Equilibrium and steady-state models of the coupling between the amino acid gradient and the sodium electrochemical gradient in mouse ascites- tumour cells
Authors: Philo R D , Eddy A A
Published: August 2015

1. The tumour cells were incubated at 37 degrees C in Ringer solutions containing glucose, 1 mM-methionine, various concentrations of Na+ and K+ and, in some instances, ouabain or valinomycin to lower the membrane potential generated by the Na+ pump. After about 30 min, when the system had reached a steady state, the ratio [extracellular Na+]/[cellular Na+] varied from about 0.6 to 3.2 with the ionic conditions. The membrane potential, determined by means of the fluorescent probe 3,3′-dipropyloxadicarbocyanine, also varied systemically from about zero to–55mV.2. the ratio [cellular methionine]/[extra-cellular methionine] varied from about 1 to 35 in these circumstances. The Na+ electrochemical gradient, measured in the same units, varied from about 1 to 30. Its magnitude in 46 assays was almost directly proportional to, though slightly smaller than, the methionine gradient. 3. A mathematical model was used to define the relation between these two gradients, which were not in equilibrium, owing to the presence of a leak pathway for the amino acid. On the assumption that the values of [cellular Na+] were correct, the methionine gradient was about 1.8 times larger than this version of the gradient hypothesis predicted.
Uptake and degradation of iodine-labelled chylomicron remnant particles by monolayers of rat hepatocytes
Authors: Florén C H , Nilsson A
Published: August 2015

1. Rat chylomicrons were labelled with 125I with 69–72% of the iodine in the protein moiety. Less than 1 nmol of iodine was incorporated per nmol of protein. Of the peptide radioactivity 44–56% was in apolipoprotein A-1, 30–40% in the C peptides and 11–15% in apolipoprotine B. The arginine-rich peptide, which accounted for about 14% of the chylomicron protein mass as determined by scanning of sodium dodecyl sulphate-polyacrylamide gels, contained very little radioactivity. 2. Chylomicron remnants generated with postheparin plasma from iodine-labelled chylomicrons showed a relative increase in the percentage of the arginine-rich peptide (76–90% of the apolipoprotein mass according to gel scanning). The major portion of the peptide iodine label was present in apolipoprotein A-1 (43–57%), B (22–32%) and C peptides (17–35%). 3. When iodine-labelled chylomicron remnants were added to rat hepatocytes in primary culture, labelled peptides were taken up and degraded by the hepatocytes by a saturable process. The Vmax. for the uptake was calculated to the 300ng of protein/h per mg of cell protein and the apparent Km as 7.7 microgram of protein/mg of cell protein. A larger proportion of the 125I-labelled lipids of the remnants (mainly polar lipids) was taken up. This suggest that these may also enter the cells by a mechanism that does not involve particulate uptake, such as phospholipid exchange. 4. The degradation of labelled peptides was inhibited by colchicine, concanavalin A, chloroquine and NH4Cl, which also inhibit degradation of the cholesteryl ester portion. All these drugs exerted their inhibition mainly after the uptake of labelled peptide. No degradation occurred at 4 degrees C, and also the uptake was markedly decreased. 5. The uptake of labelled chylomicron remnant peptide was 77 times as effective as that of labelled sucrose, which is likely to be taken up randomly by pinocytosis.
Zinc-binding protein in the livers of neonatal, normal and partially hepatectomized rats
Authors: Ohtake H , Hasegawa K , Koga M
Published: August 2015

In the livers of rats after partial hepatectomy the zinc concentration began to increase soon after the operation, reached a maximum value at 14h, and decreased to the original value by 25h after the operation. In contrast, the plasma zinc concentration continued to decrease during the first 10h after the operation and remained depressed for at least 28h. The plasma and hepatic zinc concentrations were relatively unaffected by sham-operation. Synchronous with the increase in the hepatic zinc concentration after the partial hepatectomy, there was an appearance of zinc-binding protein (Zn-binding protein) in the liver cytosol. Studies with small doses of actinomycin D and cycloheximide suggest that both RNA and protein syntheses are necessary for the induction of Zn-binding protein after partial hepatectomy. A high content of the Zn-binding protein was found in neonatal rat liver. The Zn-binding protein, however, was undetectable 40 days after birth. The Zn-binding protein was also found in the adult rat liver when stimulated to proliferate after the administration of isoprenaline followed by glucagon. These findings indicate a close linkage between the appearance of Zn-binding protein in the liver cytosol and the regulation of DNA synthesis.