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S Tanase

Publications and source records attributed to S Tanase.

87 records · Page 5Linked to original sources
PubMed1982

A critical arginine residue in cytosolic aspartate aminotransferase from pig heart.

Reaction of the pyridoxal form of cytosolic aspartate aminotransferase from pig heart with 1,2-cyclohexanedione or other alpha-dicarbonyls led to a progressive decrease in the enzymic activity toward natural dicarboxylic substrates. The inactivation was prevented by the presence of dicarboxylic substrate analogs. The dependence of the inactivation rate on the cyclohexanedione concentration indicated that the modifying reagent forms a dissociable complex with the enzyme prior to the inactivation. These saturation kinetics were observed also with other alpha-dicarbonyls tested. The inactivation was fully accounted for by the modification of a single arginine residue per monomeric unit of the enzyme. Activities for alpha, beta-elimination reaction with 3-chloro-L-alanine and transamination with L-alanine did not decrease but appeared to increase considerably with the progress of the arginine modification. In these aberrant reactions, affinity for the monocarboxylic substrates was higher with the modified enzyme than with the native unmodified enzyme. Glutamate or aspartate was still capable of reacting with the pyridoxal form of the extensively modified enzyme to produce the pyridoxamine form at a rate comparable to that of the reaction with 3-chloro-L-alanine or L-alanine. Succinate, glutarate, maleate, 2-methylaspartate or erythro-3-hydroxy-aspartate which bind strongly to the native enzyme and thus acts as potent inhibitors in the reactions with monocarboxylic substrates did not exhibit any appreciable inhibitory effect on these reactions catalyzed by the arginine-modified enzyme. Proton NMR spectroscopy demonstrated that succinate strongly interacts with the native enzyme to generate substantial changes in the enzyme spectra whereas there was no such evidence for the specific interaction with this dicarboxylate with the arginine-modified enzyme.

Animals↗
PubMed1980

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Tryptic peptides.

The amino acid sequences of 39 tryptic peptides from carboxymethylated mitochondrial aspartate aminotransferase from pig heart muscle were analyzed. The peptides were purified by gel filtration, ion exchange column chromatography, paper chromatography, and high voltage paper electrophoresis, and their sequences were examined by manual Edman degradation, carboxypeptidase digestion, and fragmentation with thermolysin or chymotrypsin. These peptides accounted for 318 of the total 401 amino acid residues in the protein subunit.

Amino Acid Sequence↗
PubMed1980

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Cyanogen bromide peptides.

Twelve cyanogen bromide peptides were isolated from S-carboxymethylated mitochondrial aspartate aminotransferase and their amino acid sequences were determined. These peptides were purified first by gel filtration on a Sephadex G-75 column, and then by gel filtration on Bio-Gel, or by ion exchange chromatography on a phosphocellulose column in the presence of 8 M urea, or by both methods. Small peptides were purified by paper chromatography. The cyanogen bromide peptides accounted for 367 of the 401 amino acid residues in the subunit of the enzyme. No peptide accounting for the other 34 residues was obtained in a homogeneous state, but peptide mixtures containing this particular peptide were analyzed by various procedures including Edman degradation and digestion with Staphylococcus aureus protease. The results accounted for all 401 amino acid residues.

Amino Acid Sequence↗
PubMed1979

Pyridoxal 5'-phosphate binding site of pig heart alanine aminotransferase.

After borohydride reduction, carboxymethylation, and tryptic digestion of the holoenzyme of pig heart alanine aminotransferase, a single icosapeptide containing the N6-(phosphopyridoxyl)lysine residue was isolated by a combination of gel filtration and ion-exchange chromatogrpahy. Its primary structure was determined as Gln-Glu-Leu-Ala-Ser-Phe-His-Ser-Val-Ser-Lsy(Pxy)-Gly-Phe-Met-Gly-Glu-Cys-Gly-Phe-Arg.

Alanine Transaminase↗
PubMed1978

Chemical structure of the active site of pig heart mitochondrial aspartate aminotransferase labeled with beta-chloro-l-alanine.

Formate-induced inactivation of pig heart mitochondrial aspartate aminotransferase by beta-chloro-L-alanine resulted in the modification of the epsilon-amino group of the lysyl residue which is involved in the formation of an aldimine bond with 4-formyl group of the coenzyme, pyridoxal 5'-phosphate. The tryptic peptide isolated from the labeled site of the enzyme was composed of 25 residues and exhibited positive circular dichroism at 325 and 254 nm where the pyridoxyl chromophore of the labeled site peptide absorbs, while the phosphopyridoxyl peptide isolated from the boro-hydride-reduced enzyme did not show any ellipticity in this spectral region. Its comparison with the analogous tryptic peptide from the labeled site of the cytosolic isoenzyme revealed a high degree of homology in their primary structures as well as in spectral properties. Structural analysis of the labeled site peptide and mechanistic consideration of the labeling process indicated that with both isoenzymes the phosphopyridoxyl group is covalently bound to the alpha amino group of the alanyl moiety derived from beta-chloro-L-alanine, the beta carbon of which is covalently linked to the epsilon-amino group of the lysyl residue.

Alanine↗
PubMed1977

The complete amino acid seqeunce of mitochondrial asparatate aminotransferase from pig heart.

The complete amino acid sequence of the mitochondrial aspartate aminotransferase from pig heart was determined by analyses of the fragments obtained from tryptic digestion and cyanogen bromide treatment of the protein. The sequence analyzer was useful for establishing the primary structure of the N-terminal portion of the whole protein. There are 401 amino acid residues in the molecule. The sequence was compared with that of the cytoplasmic isozyme, showing 48% homology.

Amino Acid Sequence↗
PubMed1977

Large-scale preparation of cytosolic and mitochondrial asparatate aminotransferases from pig heart.

A procedure is described for the large-scale preparation of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase from pig heart. The procedure consists of: 1. extraction of both isoenzymes by heat treatment of homogenates prepared from minced and frozen heat muscle; 2. separation of each isoenzyme on a hydroxyapatite column; 3. purification of each isoenzyme by combinations of heat treatment, ammonium sulfate fractionation and chromatography on ion-exchange cellulose columns. Purified preparations of each isoenzyme thus obtained were homogeneous proteins as judged from their spectral properties and behavior on polyacrylamide gel electrophoresis. Using the present procedure, 1.2 g of the cytosolic isoenzyme and 1.7 g of the mitochondrial isoenzyme were obtained from 20 kg of minced heart muscle.

Ammonium Sulfate↗
PubMedDate not supplied

Plasma clearance of intravenously injected aspartate aminotransferase isozymes: evidence for preferential uptake by sinusoidal liver cells.

Both cytosolic (c-AAT) and mitochondrial (m-AAT) isozymes of aspartate aminotransferase (EC 2.6.1.1) appear in serum in some diseases including hepatobiliary dysfunction. The present study aimed at elucidation of the mechanism by which AAT isozymes are cleared from blood. Intravenous injection into rats of m-AAT and c-AAT purified from rat liver exhibited a biphasic clearance curve with an overall half-life of 42 min and 4.7 hr, respectively. The tissue distribution of the radioactivity following intravenous administration of 125I-labeled isozymes revealed that the liver is a major organ involved in plasma clearance of these isozymes. This conclusion was also supported by the significant retardation in plasma clearance of m-AAT in hepatectomized as well as CCl4-intoxicated rats. Furthermore, clearance rate of each AAT isozyme in an isolated perfused liver exhibited a single exponential process with the uptake rate for m-AAT being much faster than that for c-AAT. Separation of hepatocytes and sinusoidal liver cells from the rat intravenously injected with 125I-labeled AAT isozymes revealed that sinusoidal cells were responsible for the plasma clearances. In vitro uptake study showed that both isozymes were exclusively taken up by sinusoidal liver cells. The uptake rate for m-AAT was considerably greater than that for c-AAT. Endocytotic index for uptake by sinusoidal cells was 16 times with c-AAT and 34 times with m-AAT as compared with that for inulin or dextran which are taken up by fluid-phase endocytosis, suggesting involvement of adsorptive endocytosis in the uptake of the isozymes.

Animals↗