scholarly journals Purification and properties of dolphin muscle aspartate and alanine transaminases and their possible roles in the energy metabolism of diving mammals

1974 ◽  
Vol 143 (3) ◽  
pp. 541-553 ◽  
Author(s):  
Terrance G. Owen ◽  
Peter W. Hochachka
Keyword(s):  
Energy Metabolism ◽  
Ph Value ◽  
Tricarboxylic Acid Cycle ◽  
Equilibrium Constants ◽  
Dicarboxylic Acids ◽  
Redox Balance ◽  
Alanine Transaminase ◽  
Starch Gel Electrophoresis ◽  
Purification And Properties ◽  
Starch Gel

1. Mitochondrial and supernatant aspartate transaminases (EC 2.6.1.1) and supernatant alanine transaminase (EC 2.6.1.2) were purified 89-, 204- and 240-fold respectively, from dolphin muscle. Starch-gel electrophoresis of crude and purified preparations revealed that all three enzymes exist as single forms. 2. Km values of α-oxoglutarate, alanine, pyruvate and glutamate for the alanine transaminase were 0.45, 8.2, 0.87 and 15mm respectively. For the aspartate transaminases, the Km values of α-oxoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4mm respectively, for the mitochondrial form and 0.13, 2.4, 0.06 and 3.2mm respectively, for the supernatant form. 3. In all cases, as the assay pH value was decreased from pH7.3, the Km values of the α-oxo acids decreased whereas those of the amino acids increased. 4. The apparent equilibrium constants for the aspartate transaminases were independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms respectively, where [Formula: see text] 5. Studies of the inhibition of the aspartate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. 6. Three key roles are suggested for the transaminases in the energy metabolism of the diving animal. First, it is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing (a) fumarate from aspartate for the ATP-producing reversal of succinate dehydrogenase, and (b) α-oxoglutarate from glutamate for the GTP-producing succinyl thiokinase reaction. Secondly, diving mammals probably accumulate more NADH than other mammals during hypoxia. The aspartate transaminases seem particularly well suited for restoring and maintaining redox balance via the malate–aspartate cycle after aerobic metabolism is resumed. Finally, since the preferred fuel for aerobic work is fat, the combined reactions of the transaminases could be instrumental in providing increased supplies of oxaloacetate for sparking the tricarboxylic acid cycle.

Purification and properties of crustacyanin

1966 ◽  
Vol 164 (994) ◽  
pp. 130-151 ◽  
Keyword(s):  
Molecular Weight ◽  
Quaternary Structure ◽  
Gel Filtration ◽  
Potassium Thiocyanate ◽  
Starch Gel Electrophoresis ◽  
Prosthetic Group ◽  
Reversible Dissociation ◽  
Purification And Properties ◽  
Starch Gel ◽  
Quaternary Structures

Crustacyanin, the blue carapace pigment of the common lobster Homarus gammarus (L.), has been purified and crystallized. This chromoprotein has a minimum molecular weight of 36 000 based on the content of the carotenoid prosthetic group astaxanthin. The molecular weight in gel filtration measurements is about 650 000, corresponding to some 18 molecules of astaxanthin per molecule of protein. Crustacyanin, on dialysis against water, dissociates into particles of about 35 000 molecular weight, each apparently bearing one molecule of carotenoid. The dissociation is accompanied by a shift in the principal maximum of the absorption spectrum from 633 to 595 nm and is reversed upon addition of salt. Reversible dissociation also occurs in the presence of 3 M urea, 1 M potassium thiocyanate, 10% (v/v) dioxan or 10% (v/v) acetone. When the carotenoid is removed from crustacyanin with acetone, the resul­tant apoprotein has a mean molecular weight of about 20 000. It may be resolved by starch gel electrophoresis into several components of which two predominate. Crustacyanin, indistinguishable from the native material, can be reconstituted from apoprotein and carotenoid. Evidence from the behaviour of crustacyanin and its apoprotein at surfaces indicates that the tertiary and quaternary structures of the native protein are stabilized by the carotenoid. It is suggested that the quaternary structure of crustacyanin is induced by an interaction of the carotenoid molecules of the subunits, which in turn causes a change in configuration of the protein favourable to aggregation. The result is a micelle-like structure with a hydrophobic carotenoid core.

scholarly journals Purification and properties of esterases characteristic of adult rat brain

1968 ◽  
Vol 110 (4) ◽  
pp. 713-719 ◽  
Author(s):  
Danica Dabich ◽  
Bhargavan Chakrapani ◽  
Frank N. Syner
Keyword(s):  
Partial Purification ◽  
Starch Gel Electrophoresis ◽  
Newborn Rat ◽  
Cholesteryl Acetate ◽  
Adult Rat ◽  
Purification And Properties ◽  
Adult Rat Brain ◽  
Starch Gel ◽  
The Brain ◽  
Naphthyl Acetate

1. A method for the partial purification of an esterase fraction, present in the brain of the adult but not the newborn rat, is described. A 54-fold purification was achieved in three steps. 2. When subjected to starch-gel electrophoresis, the purified fraction resolved into three bands of esterase activity. Two of these bands migrate close together and faster than other esterases in the brain. These two esterases are inhibited by p-hydroxymercuribenzoate but not by di-isopropyl phosphorofluoridate. The third band is di-isopropyl phosphorofluoridate-sensitive and migrates just behind the two leading esterases. 3. After treatment with di-isopropyl phosphorofluoridate, to obviate the effects of the di-isopropyl phosphorofluoridate-sensitive esterase, the enzyme preparation hydrolyses α-naphthyl acetate, α-naphthyl propionate and α-naphthyl butyrate, but not cholesteryl acetate. The Vmax. for the naphthyl esters decreased with increase in chain length of the acyl group. The acetate ester is hydrolysed 34 times as fast as the butyrate and about seven times as fast as the propionate derivative. The Km values for these three esters, measured at pH7·2 and 37°, are 2·8×10−4m, 3·1×10−4m and 7·3×10−5m for the acetate, propionate and butyrate derivatives respectively. 4. The Hofstee (1952) plots for the kinetic data show a single line, indicating that the two most-rapidly migrating esterases, although electrophoretically separable, are not kinetically distinguishable in the substrate ranges examined.

Bovine Platelet Proteins II. Purification of Platelet Fibrinogen

1969 ◽  
Vol 21 (03) ◽  
pp. 419-427 ◽  
Author(s):  
N. O Solum ◽  
S Łopaciuk
Keyword(s):  
Analytical Ultracentrifugation ◽  
Insoluble Fraction ◽  
Disc Electrophoresis ◽  
Plasma Fibrinogen ◽  
Starch Gel Electrophoresis ◽  
Freezing And Thawing ◽  
Insoluble Material ◽  
High Resolution Power ◽  
Starch Gel ◽  
Platelet Proteins

Summary1. Platelet fibrinogen has been purified from washed bovine platelets. The procedure was based on the methods for purification of plasma fibrinogen by fractionated precipitations and extractions with ethanol and glycine below 0°, and precipitation of proteins by dimethylformamide at 0°.2. The platelet extract obtained by freezing and thawing of the cells, freed from insoluble material by centrifugation at 23,000 x g for 30 min, contained 0.22 ±0.003mg fibrinogen per 109 platelets. Total protein of this fraction was 0.77 ±0.08 mg per 109 platelets whereas that of the insoluble fraction was 0.79 ±0.09 mg per 109 platelets.3. The most purified platelet fibrinogen fraction contained 91-98% of the protein in a thrombin-clottable state. The yield was approx. 20%. It showed homogeneity in analytical ultracentrifugation, in immunoelectrophoresis using an antiserum produced by immunization of rabbits against platelet extract, and in starch gel electrophoresis using a discontinuous system of Tris HCl and borate buffers offering a high resolution power towards the platelet proteins. Polyacrylamide disc electrophoresis revealed two to three faint lines behind the main fibrinogen line. At least one such line was also observed with purified plasma fibrinogen.

Fractionation of Plasminogen by Starch Gel Electrophoresis

1964 ◽  
Vol 12 (01) ◽  
pp. 126-136 ◽  
Author(s):  
Karl H. Slotta ◽  
J. D Gonzalez
Keyword(s):  
Gel Electrophoresis ◽  
Room Temperature ◽  
Broad Band ◽  
Caproic Acid ◽  
Starch Gel Electrophoresis ◽  
Full Activity ◽  
Veronal Buffer ◽  
Starch Gel ◽  
Alkaline Range

SummaryWhen urea or ε-amino caproic acid were used as solublizing agents for plasminogen in electrophoretic experiments, only one broad band of the proenzyme was obtained on acetate cellulose, in starch block, and in acrylamide gel. In starch gel electrophoresis, however, both forms of plasminogen – the native or euglobulin and Kline’s or Pseudoglobulin plasminogen – separated into six bands. These migrated toward the cathode at room temperature in borate or veronal buffer in the alkaline range and showed full activity in fibrinagar-streptokinase plates.

scholarly journals PROTEIN POLYMORPHISMS, SEGREGATION IN GENETIC CROSSES AND GENETIC DISTANCES AMONG FISHES OF THE GENUS XIPHOPHORUS (POECILIIDAE)

Genetics ◽  
1982 ◽  
Vol 102 (3) ◽  
pp. 539-556
Author(s):  
Don C Morizot ◽  
Michael J Siciliano
Keyword(s):  
Goodness Of Fit ◽  
Inbred Strains ◽  
Interspecific Backcross ◽  
Genetic Distances ◽  
Rapid Expansion ◽  
Starch Gel Electrophoresis ◽  
Lower Vertebrate ◽  
Protein Coding ◽  
Group I ◽  
Starch Gel

ABSTRACT The products of 49 protein-coding loci were examined by starch gel electrophoresis for populational variation in six species of Xiphophorus fishes and/or segregation in intra- and interspecific backcross and intercross hybrids. Electrophoretic variation was observed for 29 of the 35 locus products in a survey of 42 population samples. The highest frequency of polymorphic loci observed in noninbred populations was 0.143. After ten or more generations of inbreeding, all loci studied were monomorphic. Inbred strains generally exhibited the commonest electrophoretic alleles of the population from which they were derived. An assessment of genetic distances among Xiphophorus populations reflected classical systematic relationships and suggested incipient subspeciation between X. maculatus from different drainages as well as several species groups. Thirty-three loci were analyzed with respect to segregation in hybrids. The goodness of fit of segregations to Mendelian expectations at all loci analyzed (except loci in linkage group I) is interpreted as evidence for high genetic compatibility of the genomes of Xiphophorus species. It is anticipated that these data will result in a rapid expansion of the assignment of protein-coding loci to linkage groups in these lower vertebrate species.

scholarly journals THE INHERITANCE OF ENZYME VARIANTS FOR TYROSINE AMINOTRANSFERASE, NADP-DEPENDENT MALATE DEHYDROGENASE, NADP-DEPENDENT ISOCITRATE DEHYDROGENASE, AND TETRAZOLIUM OXIDASE IN TETRAHYMENA PYRIFORMIS, SYNGEN 1

Genetics ◽  
1973 ◽  
Vol 74 (4) ◽  
pp. 595-603
Author(s):  
D Borden ◽  
E T Miller ◽  
D L Nanney ◽  
G S Whitt
Keyword(s):  
Detailed Analysis ◽  
Malate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Tetrahymena Pyriformis ◽  
Tyrosine Aminotransferase ◽  
Breeding Program ◽  
Starch Gel Electrophoresis ◽  
Enzyme Locus ◽  
Starch Gel

ABSTRACT The isozymic patterns of tyrosine aminotransferase, NADP malate dehydrogenase, NADP isocitrate dehydrogenase, and tetrazolium oxidase were examined by starch-gel electrophoresis in Tetrahymena pyriformis, syngen 1. The genetics of the alleles controlling these enzymes was studied through a breeding program. Each enzyme locus was shown to assort vegetatively, as do other loci in this organism. A detailed analysis of the assortment process for the tyrosine aminotransferase locus indicated that the rate of stabilization of heterozygotes into pure types was essentially identical to previously-reported rates for other loci.

scholarly journals Malonyl-proteome profiles of Staphylococcus aureus reveal lysine malonylation modification in enzymes involved in energy metabolism

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yanan Shi ◽  
Jingjing Zhu ◽  
Yan Xu ◽  
Xiaozhao Tang ◽  
Zushun Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Energy Metabolism ◽  
Active Sites ◽  
Current Knowledge ◽  
Tricarboxylic Acid Cycle ◽  
Protein A ◽  
Pentose Phosphate ◽  
Post Translational Modification ◽  
Bacterial Physiology ◽  
Dihydrolipoyl Dehydrogenase

Abstract Background Protein lysine malonylation, a novel post-translational modification (PTM), has been recently linked with energy metabolism in bacteria. Staphylococcus aureus is the third most important foodborne pathogen worldwide. Nonetheless, substrates and biological roles of malonylation are still poorly understood in this pathogen. Results Using anti-malonyl-lysine antibody enrichment and high-resolution LC-MS/MS analysis, 440 lysine-malonylated sites were identified in 281 proteins of S. aureus strain. The frequency of valine in position − 1 and alanine at + 2 and + 4 positions was high. KEGG pathway analysis showed that six categories were highly enriched, including ribosome, glycolysis/gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), valine, leucine, isoleucine degradation, and aminoacyl-tRNA biosynthesis. In total, 31 malonylated sites in S. aureus shared homology with lysine-malonylated sites previously identified in E. coli, indicating malonylated proteins are highly conserved among bacteria. Key rate-limiting enzymes in central carbon metabolic pathways were also found to be malonylated in S. aureus, namely pyruvate kinase (PYK), 6-phosphofructokinase, phosphoglycerate kinase, dihydrolipoyl dehydrogenase, and F1F0-ATP synthase. Notably, malonylation sites were found at or near protein active sites, including KH domain protein, thioredoxin, alanine dehydrogenase (ALD), dihydrolipoyl dehydrogenase (LpdA), pyruvate oxidase CidC, and catabolite control protein A (CcpA), thus suggesting that lysine malonylation may affect the activity of such enzymes. Conclusions Data presented herein expand the current knowledge on lysine malonylation in prokaryotes and indicate the potential roles of protein malonylation in bacterial physiology and metabolism.

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