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1.
Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with Km values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free-SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a Km of 0.057 mM. 相似文献
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Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase. 相似文献
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Activities of the alanine aminotransferase were measured along the entire sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats using a Lowry technique. The established profiles of enzyme activity give support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. 相似文献
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Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver
Maulik N 《Molecular and cellular biochemistry》2004,264(1-2):13-23
Despite significant advances in myocardial revascularization and reperfusion, coronary artery disease and subsequently myocardial infarction, are the leading causes of morbidity and mortality in the United States. Strategies which improve the myocardial substrate during and following a myocardial infarction-such as the regrowth of functional blood vessels to the ischemic myocardium would be of great clinical importance. This review article attempts to address this important clinical issue through identifying potential signalling mechanisms by various mode of preconditioning that cause angiogenesis. 相似文献
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Summary Activities of the alanine aminotransferase were measured along the entire sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats using a Lowry technique. The established profiles of enzyme activity give support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.Dedicated to Professor Dr. T.H. Schiebler on occasion of his 65th birthdaySupported by grants of the Forschungsförderung des Landes Nordrhein-Westfalen, No. 40002585 and the Verein der Förderer und Freunded der Universität Köln 相似文献
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A lipolytic zinc-copper protein has been isolated from the cytosol of chick liver. This material had a molecular weight of 6000 daltons, contained four atoms of zinc and one atom of copper per molecule. The 6000 dalton fraction aggregated at high ionic strength or in the presence of sodium dodecyl sulphate. Lipolytic activity was observed towards triolein, tripalmitin, phosphatidyl choline and retinyl palmitate, and was stimulated by cholate, Ca and high NaCl concentrations, and was inhibited by sulphydryl reagents, inhibitors of serine esterases, alkaline phosphatase and chelating agents. It appears that this copper-zinc protein is distinct from metallothionein which has no lipolytic activity. 相似文献
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The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase. 相似文献
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An affinity chromotography resin highly specific for rat liver tyrosine aminotransferase (EC 2.6.1.5) has been synthesized and used in the purification of this enzyme. The structure of the resin, N-(5′-phosphopyridoxyl)-l-tyrosyl-aminoocytl-Sepharose 4B, was designed to resemble the tyrosine-pyridoxal phosphate Schiff's base intermediate in the reaction pathway catalyzed by this enzyme. Use of this resin in combination with octyl-agarose chromatography on partially purified enzyme resulted in a tyrosine aminotransferase preparation with a specific activity of about 450 units/mg protein. When analyzed on one-dimensional polyacrylamide-sodium dodecyl sulfate slab gels, the highly purified enzyme was composed of two polypeptides with molecular weights of about 56,000 and 53,000. Radioiodinated tryptic peptides from each of these polypeptides were essentially identical following two-dimensional analysis. Although the two polypeptides could not be separated from each other in an active form, it was found that (i) both polypeptides have pyridoxal phosphate-binding sites, (ii) the coenzyme is probably bound to both polypeptides as a Schiff's base, (iii) both polypeptides have binding sites for l-tyrosine and l-glutamic acid, the two specific substrates for the enzyme, and (iv) both polypeptides can catalyze the formation of the initial amino acid-pyridoxal phosphate Schiff's base adduct in the overall reaction pathway. Since the ratios of these polypeptides differed from preparation to preparation of purified enzyme, the 53,000 Mr species probably arises by proteolysis of tyrosine aminotransferase in crude liver extracts. These results imply that if tyrosine aminotransferase isozymes exist, they are not the result of translation products produced by different structural genes. 相似文献
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A Samuni C M Krishna P Riesz E Finkelstein A Russo 《The Journal of biological chemistry》1988,263(34):17921-17924
2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes. 相似文献
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Nishijima S Sugaya K Morozumi M Hatano T Ogawa Y 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,780(1):13-19
We measured hepatic alanine:glyoxylate aminotransferase (AGT) activity using capillary electrophoresis. After rat liver homogenate was incubated in the presence of substrates and pyridoxal 5'-phosphate, the pyruvate and glycine produced by AGT were measured. The AGT activity was 10.02+/-0.31 micro mol pyruvate/h/mg protein and 10.21+/-0.15 micro mol glycine/h/mg protein. This method is relatively simple and shows superior sensitivity, allowing the measurement of enzyme activity in 5 micro g of protein. Therefore, it appears to be suitable for laboratory use and may also have advantages for measuring AGT activity in liver biopsy specimens. 相似文献
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We have identified two different species of inhibitors of calmodulin-dependent cAMP phosphodiesterase: 1) a low molecular weight (LMW) and 2) a high molecular weight (HMW) form. These inhibitors are extracted from rat liver. Both LMW and HMW inhibitors are heat-stable, acidic in nature and lose activity with prolonged storage and/or repeated freezing and thawing. The low molecular weight inhibitor has been purified to about 7,000-fold with 300% recovery. LMW inhibits calmodulin-dependent cAMP phosphodiesterase regardless of the source of calmodulin (e.g. fat, brain, heart, erythrocytes). LMW appears to be lipid in nature with a molecular weight of 1,500-5,000. The role of these inhibitors in diabetes and mechanism of action of insulin is presented. 相似文献
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Calciphorin, the putative mitochondrial calcium ionophore from rat liver mitochondria, exhibits the inherent properties of the mitochondrial calcium transport system and is similar to the calf heart preparation reported earlier. The protein has a strong selectivity for Ca2+, and has a Kd for Ca2+ of 56.5 +/- 6.6 microM and 13.9 +/- 2.1 microM in organic extraction and flow dialysis experiments, respectively. Reduction of the contaminating lipids from 23 +/- 6.5 to 1.73 +/- 0.4 moles per mole protein does not alter the affinities, Ca2+/protein stoichiometry or selectivity for Ca2+. 相似文献
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Simkhada JR Lee HJ Jang SY Kim JH Lee HC Sohng JK Yoo JC 《Bioresource technology》2009,100(3):1388-1393
With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids. 相似文献
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The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library 总被引:2,自引:0,他引:2
A novel low Mr GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000-17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras-related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H-ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells. 相似文献
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Marisa Montemartini Jacqueline Búa Esteban Bontempi Cecilia Zelada rés M. Ruiz JoséA. Santomé Juan JoséCazzulo Cristina Nowicki 《FEMS microbiology letters》1995,133(1-2):17-20
Abstract Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-trans-ferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent K m values similar to those for the natural enzyme, thus proving that they belong to the same protein. 相似文献