Affinity purification and properties of rat liver mitochondrial L-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin

The present study describes a new rapid procedure for purification of L-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The Km values for L-alanine and 4,5-dioxovalerate were 3.3 and 0.28 mM, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.     

Localization of L-alanine: 4,5-dioxovalerate transaminase in the matrix of the rat kidney mitochondria

The bulk of the enzyme L-alanine: 4,5-dioxovalerate transaminase, which catalyses the transamination reaction between L-alanine and 4,5-dioxovalerate to synthesize delta-aminolevulinic acid was predominantly recovered in the mitochondrial matrix. Sub-fractionation procedure of the mitochondria involved the use of digitonin and lubrol followed by differential centrifugation to separate soluble and particulate enzymes. Lubrol did not inhibit this enzyme. Presence of this enzyme in the mitochondrial matrix was further confirmed by western blot analysis. The results support the conclusion that L-alanine: 4,5-dioxovalerate transaminase is localized and functions in the mitochondrial matrix.     

Evidence of hemin as an end product inhibitor of L-alanine: 4,5-dioxovalerate transaminase in rat liver mitochondria

This study describes the in vitro and in vivo effect of hemin on L-alanine:4,5-dioxovalerate transaminase activity. Hemin was shown to be an inhibitor of the purified enzyme and this inhibition was proportional to the concentration of hemin. The examined kinetic data with hemin showed uncompetitive inhibition for both alanine and 4,5-dioxovalerate. An apparent Ki of 30 and 42 microM for hemin were obtained with both alanine and 4,5-dioxovalerate, respectively. Moreover, the enzyme activity in liver was considerably decreased after the intravenous hemin administration and such an inhibition is dose and time dependent. Furthermore, maximum inhibition of the enzyme was observed 30 min after hemin injection and 60% enzyme inhibition was achieved with a dose of 1.2 mg/kg body wt of rat. Thus is suggests the important role of this enzyme on heme biosynthesis.     

Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic L-alanine: 4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial L-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of L-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial L-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of L-alanine: 4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment.     

Excess generation of endogenous heme inhibits L-alanine:4,5-dioxovalerate transaminase in rat liver mitochondria

In the present study, we examined the possibility that the excess heme generation within mitochondria may provide a local concentration, sufficient to inhibit the activity of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid biosynthesis in mammalian system. This was accomplished by assaying together L-alanine:4,5-dioxovalerate transaminase and heme synthetase activities in intact mitochondria isolated from rat liver. Endogenous heme in intact mitochondria has been generated in excess, by increasing the concentration of the substrate of heme synthetase. Our studies showed that the activity of L-alanine:4,5-dioxovalerate transaminase decreased as the rate of heme formation increased. In intact mitochondria, almost 50% inhibition of alanine:4,5-dioxovalerate transaminase was obtained with 4.0 mumole of heme generation. We conclude that end product inhibition of L-alanine:4,5-dioxovalerate transaminase by hemin, which was proposed in earlier report by us (FEBS Letter (1985), 189, 129), is an important physiological mechanism for the regulation of hepatic heme biosynthesis.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.     

Purification and some properties of L-alanine:4,5-dioxovaleric acid transaminase from rat liver mitochondria

L-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.44) has been purified to homogeneity from rat liver mitochondria. Molecular weight of the native enzyme is estimated to be 230,000 +/- 3000 by gel filtration. Under denaturing condition, the dissociated enzyme has a subunit of approximately 41,000 +/- 2000, indicating the enzyme apparently is composed of six identical subunits. The enzyme is heat stable and has optimal activity at pH 6.9. Km values for L-alanine and 4,5-dioxovalerate are 3.3 X 10(-3) M and 2.8 X 10(-4) M respectively. Excess dioxovalerate inhibits the enzyme activity. Pyridoxal phosphate and dithiothreitol also inhibit the enzyme activity.     

Role of L-alanine: 4,5-dioxovalerate transaminase in chlorophyll synthesis in Bajra (Pennisetum typhoideum) seedlings

L-alanine:4,5-dioxovalerate transaminase activity and chlorophyll levels were estimated in lead and mercury treated Bajra seedlings. The enzyme activity increased with age upto 2nd day of germination and decreased on consequent days, where as the chlorophyll content increased with age upto 4th day and remained constant on day 5. Both the metals have no effect on L-alanine: 4,5-dioxovalerate transaminase activity but reduced chlorophyll levels. In vitro incubation of the enzyme with metal solutions showed that the enzyme activity was inhibited by mercury, while lead had no effect. Studies on sub-cellular localization of the L-alanine:4,5-dioxovalerate transaminase showed that it is present in all fractions. The non-correlation between L-alanine: 4,5-dioxovalerate transaminase activity and chlorophyll synthesis is evident from different activity profiles with age and response to heavy metal treatment in the seedlings. Hence, our results suggest the non-involvement of L-alanine:4,5-dioxovalerate transaminase in chlorophyll synthesis in bajra seedlings.     

L-Alanine: 4,5-dioxovalerate transaminase does not regulate heme synthesis in rat liver

L-Alanine: 4,5-dioxovalerate transaminase (ADT) was determined in liver homogenates of rats treated by either inducers of porphyrin synthesis or the repressor, hemin. ADT activity was not induced by the porphyrinogenic agents nor reduced by hemin, indicating that ADT probably has no regulatory role in the heme synthesis pathway. The same conclusion was drawn from similar experiments performed in monolayers of chick embryo liver cells.     

L-alanine: 4,5-dioxovalerate aminotransferase from Pseudomonas riboflavina: purification and inactivation by methylglyoxal

L-Alanine:4,5-dioxovalerate aminotransferase, which catalyzes transamination between L-alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from Pseudomonas riboflavina IFO 3140. The enzyme had a molecular weight of 190,000 and consisted of four identical subunits. It was crystallized as pale yellow needles. The enzyme used L-alanine (relative activity 100), beta-alanine (39), and L-ornithine (14) as amino donors. gamma-aminobutyrate (55) and epsilon-aminocaproate (34) were also effective as amino donors. The reaction proceeded according to a ping-pong mechanism and the Km values for L-alanine and 4,5-dioxovalerate were 1.7 and 0.75 mM, respectively. The activity of the enzyme is strongly inhibited by pyruvate, hemin, and methylglyoxal. Methylglyoxal interacted with the enzyme and brought about a complete inactivation.     

Purification and partial characterization of cysteine-glutamate transaminase from rat liver.

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and alpha-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of alpha-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.     

Purification and characterization of galactocerebroside sulfotransferase from rat kidney

Galactocerebroside sulfotransferase (EC 2.8.2.11) was purified to apparent homogeneity from rat kidneys. The purified protein is stable at -20 degrees C, and has an estimated molecular weight of 64,000 and a pI of 5.1. In contrast to other known sulfotransferases, the enzyme appears not to require divalent metal ions for activity. The Km for the donor, 3'-phosphoadenosine 5'-phosphosulfate, is 5.2 microM. Structural studies on this "active" sulfate donor show the requirement of a phosphate group at the 3' position of the ribose moiety. Modification of the amino group at either the 6 or 8 position on the purine ring renders the corresponding compounds poor substrates. Both galactosylceramide and lactosylceramide are effective acceptors for this enzyme, while galactosylsphingosine and galactosylglycerolipids are sulfated only poorly, suggesting that the in vivo sulfation of these glycolipids is carried out by different sulfotransferases. The active site of the enzyme contains arginine residues which appear to be important in binding the sulfate donor. The enzyme protein is hydrophobic and binds 0.17 mg [3H]Triton X-100/mg protein. The purified enzyme contains bound lipids, consisting primarily of cholesterol and phosphatidylcholine. The lipid environment affects the activity of the enzyme which, in turn, regulates the sulfation of glycolipids.     

Purification and characterization of fatty acid-binding protein from rat kidney

We detected the presence of a fatty acid-binding protein (FABP) in rat kidney cytosols. This protein was eluted and purified 9.3-fold by sequential gel filtration and anion-exchange chromatography. Homogeneity was shown by a single band on polyacrylamide gel with a molecular weight of about 15,500. It had an optimum binding pH of 7.4. The binding of palmitate to the protein was saturable. Examination of fatty acid binding revealed the presence of a single class of fatty acid-binding sites. The apparent dissociation constant was 1.0 microM and the maximal binding capacity was 48 nmol/mg of protein. This protein showed similar binding characteristics for palmitate, oleate, and arachidonate. Rabbit antibody to this cytosolic FABP gave a single precipitin line with the antigen and selectively inhibited [14C]palmitate binding to the protein.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

Purification and characterization of the activated mineralocorticoid receptor from rat kidney

1. The mineralcorticoid receptor (MR) from rat kidney was purified within 8 hr by the following, successive steps: stabilization with synthetic, tritiated steroids (RU 26752 or R 5020), phosphocellulose passage, heat activation (25 degrees C), and DNA-cellulose batch elution. 2. The purified preparation was resolved as a single, 75 KDa band on SDS-PAGE electrophoresis although the exact degree of purity was difficult to assess by the charcoal assay due to denaturation. 3. The natural hormone, aldosterone, was unsuitable for receptor purification and characterization. 4. The MR purified with different ligands behaved identically during ion exchange and gel permeation analyses, suggesting post-translational modifications of the native receptor in whole cytosol that exhibits molecular heterogeneity.