Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

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.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

A novel low molecular weight alanine aminotransferase from fasted rat liver

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.     

A novel low molecular weight alanine aminotransferase from fasted rat liver

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 K_m 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 K_m of 0.057 mM.     

Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

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.     

Distribution pattern of alanine aminotransferase activity in rat liver

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.     

Distribution pattern of alanine aminotransferase activity in rat liver

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     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Isolation and characterization of rat kidney alanine aminopeptidase

Rat alanine aminopeptidase was purified from kidney by isolation of the brush border membrane with CaCl2 followed by differential centrifugation and tryptic proteolysis. It is a glycoprotein with a molecular weight of approximately 210,000 daltons comprising two 110,000-dalton subunits and has an amino acid composition similar to that of the human enzyme. Two zinc atoms are covalently bound to each protein subunit.     

Purification and characterization of alanine aminotransferase from Panicum miliaceum leaves

Three alanine aminotransferases, two minor (AlaAT-1, AlaAT-3) and one major (AlaAT-2), were detected by native gel electrophoresis of leaf extracts from Panicum miliaceum L. AlaAT-2 was purified to homogeneity and a specific polyclonal antibody was raised against it which did not react with the other two forms of the enzyme. The enzyme, with an apparent molecular size of 102 kDa, appeared to be a dimer of a single 50-kDa polypeptide. The enzyme has a relatively broad pH optima with similar curves for the forward and reverse directions, ranging between 6.5 and 7.5. The Km values of this enzyme were 6.67, 0.15, 5.00, and 0.33 mM for alanine, 2-oxoglutarate, glutamate, and pyruvate, respectively. The activity of AlaAT-2 was found to increase markedly during leaf greening in parallel with the increase of immunochemically titrated protein, and it is suggested to function in the C4 photosynthetic cycle.     

Isolation and characterization of deteriosomes from rat liver

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269–2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 μm to 0.11 μm in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A₂ activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A₂ is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.     

Purification and characterization of D-3-aminoisobutyrate-pyruvate aminotransferase from rat liver

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) was purified 1900-fold from rat liver extract. The purified enzyme showed a molecular mass of 180 kDa by gel-permeation HPLC analysis using a TSK gel G3000SW column. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in identification of a single band of approx. 50 kDa, indicating that the native enzyme is probably a tetrametric protein. The specific activity of the purified enzyme was 1.14 mumol/min per mg protein. D-3-Aminoisobutyrate and beta-alanine were good amino donors. The Km value for L-3-aminoisobutyrate was 100-times larger than that for the D-isomer. The apparent Km values for D-3-aminoisobutyrate and beta-alanine were 35 and 282 microM, respectively. Pyruvate, glyoxylate, oxalacetate, 2-oxo-n-valerate, and 2-oxo-n-butyrate were good amino acceptors. The apparent Km values for pyruvate and glyoxylate were 32 and 44 microM, respectively.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita

Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for l-alanine were 18.5+/-0.48 and 23.55+/-0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29+/-0.023 and 0.33+/-0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With l-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4 x 10(-4) and 3.4 x 10(-4) mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate l-alanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands.     

Purification and characterization of aromatic-amino-acid-glyoxylate aminotransferase from monkey and rat liver.

Aromatic-amino-acid-glyoxylate aminotransferase was highly purified from the mitochondrial fraction of livers from monkey and glucagon-injected rats. The two enzyme preparations showed physical and enzymic properties different from a kynurenine aminotransferase previously described. The two enzymes had nearly identical molecular weights (approximate 80 000), isoelectric points (pH 8.0) and pH optima (pH 8.0 - 8.5). However, a difference in substrate specificity was observed between the two enzymes. Both enzymes utilized glyoxylate, pyruvate, hydroxypyruvate and 2-oxo-4-methyl-thiobutyrate as effective amino acceptors. 2-Oxoglutarate was active for rat enzyme but not for monkey enzyme. With glyoxylate, amino donors were effective in the following order of activity; phenylalanine greater than histidine greater than tyrosine greater than tryptophan greater than 5-hydroxytrypotphan greater than kynurenine for the rat enzyme, and phenylalanine greater than kynurenine greater than histidine greater than tryptophan greater than 5-hydroxy-tryptophan for the monkey enzyme.     

Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.