Degradation of phenylalanine:pyruvate transaminase after glucagon treatment

Using a single-isotope and immune precipitation technique the half-life (t 1/2) of hepatic phenylalanine:pyruvate transaminase (aminotransferase, EC 2.6.1.--, Number not yet assigned) from glucagon-treated rats was determined to be 2.8 days, similar to that of the control rats (t 1/2 = 3.3 days). The half-life of rat liver total soluble proteins also remained unchanged after glucagon treatment (t 1/2 = 2.7 days in glucagon-treated rats; t 1/2 = 2.8 days in normal). Thus, glucagon has no effect on the degradation of phenylalanine:pyruvate transaminase. Furthermore, the degradation rates are similar for both the holoenzyme and the apoenzyme of phenylalanine:pyruvate transaminase.     

Direct evidence for de novo synthesis of rat liver phenylalanine: pyruvate transaminase after glucagon treatment.

A specific antibody to phenylalanine:pyruvate transaminase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by glucagon and N⁶,O^2′-dibutyryl cyclic AMP. Cycloheximide given simultaneously with glucagon or dibutyryl cyclic AMP blocked the increase in [³H]leucine incorporation when it was injected along with glucagon, but had no effect when given 4 h after the glucagon. This finding suggests that the mRNA synthesis for phenylalanine:pyruvate transaminase may be completed in 4 h.     

Species distribution and properties of hepatic phenylalanine (histidine):pyruvate aminotransferase.

Hepatic phenylalanine(histidine):pyruvate aminotransferase activity is much higher in the mouse and rat than in other animal species (human, guinea-pig, rabbit, pig, dog and chicken). The activity is elevated in the mouse and rat by the injection of glucagon but not in other species (guinea-pig, rabbit and chicken). The enzyme was purified from the mitochondrial fraction of mouse liver to homogeneity as judged by polyacrylamide disc gel electrophoresis in the presence of dodecylsulphate. With histidine as amino donor, the enzyme was active with pyruvate, oxaloacetate and hydroxypyruvate as amino acceptors but not with 2-oxoglutarate. Effective amino donors were histidine, phenylalanine and tyrosine with pyruvate, and methionine, serine and glutamine with phenylpyruvate. The apparent Km for histidine was about 6.9 mM with pyruvate and that for pyruvate was 21 mM with histidine. The enzyme is probably composed of two identical subunits with a molecular weight of approximately 40000. The pH optimum was near 9.0. Isoelectric focusing of the purified enzyme resulted in the detection of four forms with pI 6.0, 6.2, 6.5 and 6.7, respectively, all of which were responsive to glucagon. These four forms were nearly identical with the purified enzyme before the focusing with respect to physical and enzymic properties. A possible mechanism of this multiplicity is discussed.     

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.     

Purification and properties of two forms of rat alpha-lactalbumin.

Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by the DEAE-cellulose chromatography. alpha-Lactalbumin1 is a bigger molecule in contrast to other known alpha-lactalbumins, and has a molecular weight of 21,500 as determined by sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel analysis. alpha-Lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. alpha-Lactalbumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.     

Purification and characterization of rat liver mitochondrial phenylalanine pyruvate aminotransferase.

Phenylalanine pyruvate aminotransferase in rat liver was found in both the mitochondrial and supernatant fractions. Phenylalanine pyruvate aminotransferase was purified from rat liver mitochondria. The purified enzyme was specific for pyruvate, exhibiting no activity with 2-oxoglutarate as aminoacceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order of activity: L-phenylalanine > L-tyrosine > L-histidine > 3,4-dihydroxy-DL-phenylalanine. Very little activity was observed with L-tryptophan and 5-hydroxy-L-tryptophan. The apparent Km values for L-phenylalanine and L-histidine were 2.6 mM and 2.7 mM, respectively. The Km values for pyruvate were 5.0 mM and 1.5 mM with phenylalanine and histidine as amino donors, respectively. The pH optimum was near 9.0. Sucrose density gradient centrifugation gave a molecular weight of approximately 68,000. On the basis of subcellular distributions, substrate specificities, substrate inhibition, pH optima, polyacrylamide gel electrophoresis and some other properties, it was suggested that mitochondrial phenylalanine pyruvate aminotransferase was identical with mitochondrial histidine pyruvate aminotransferase.     

Purification and properties of pyruvate kinase from Streptococcus mutans.

Pyruvate kinase (EC 2.7.1.40) from Streptococcus mutans strain JC2 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 180,000 to 190,000, and the enzyme was considered to consist of four identical subunits. This enzyme was completely dependent on glucose 6-phosphate for activity, and the saturation curve for activation by glucose 6-phosphate was sigmoidal. In the presence of 0.5 mM glucose 6-phosphate, the saturation curves for the substrates phosphoenolpyruvate and ADP were hyperbolic, and the Km values were 0.22 and 0.39 mM, respectively. GDP, IDP, and UDP could replace ADP, and the Km for GDP (0.026 mM) was 0.067 of that for ADP. The enzyme required not only divalent cations, Mg2+ or Mn2+, but also monovalent cations, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for glucose 6-phosphate increased. However, the enzyme was immediately inactivated in a solution without Pi. The intracellular concentration of glucose 6-phosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. mutans.     

Purification and properties of hydroxypyruvate: l-Alanine transaminase from rabbit liver

A procedure is described for the extensive purification of hydroxypyruvate:l-alanine transaminase from rabbit liver. On the basis of gel filtration studies, the molecular weight of the enzyme is estimated to be about 41,000 daltons. A similar value was obtained when the enzyme was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate indicating that the enzyme consists of a single polypeptide chain.The purified enzyme catalyzes the transamination of glyoxylate as well as hydroxypyruvate with l-alanine as the preferred amino donor for both substrates. The two enzymatic activities were not separated during purification nor by Chromatographic or electrophoretic procedures. Kinetic studies demonstrated that the two α-keto acids are competitive substrates. The above data are consistent with the fact that a single enzyme catalyzes the transamination of both glyoxylate and hydroxypyruvate. The effects of various inhibitors on enzymatic activity were investigated. The enzyme is inhibited by glyceraldehyde-3-phosphate and other aldehydes.The possible role of hydroxypyruvate:l-alanine transaminase in gluconeogenesis is discussed.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.     

Effect of glucagon on hepatic phenylalanine hydroxylase in vivo

Moderate doses of glucagon (20 g/kg I.V.) are sufficient to stimulate rat hepatic phenylalanine hydroxylase in vivo. In addition, the stimulation of the tetrahydrobiopterin-dependent phenylalanine hydroxylase activity in livers of animals fed on a high-protein diet has been correlated with an elevated phosphate content. The tetrahydrobiopterin-dependent hydroxylase activity in these animals can be further elevated by glucagon-stimulated phosphorylation. These results indicate that physiological changes in glucagon concentration modulate rat liver phenylalanine hydroxylase activity in vivo. The current understanding of the role of phosphorylation in regulating human phenylalanine hydroxylase is also considered.     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.     

Purification and properties of two multiple forms of dihydrodiol dehydrogenase from guinea-pig testis

NADP+-dependent dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol: NADP+ oxidoreductase, EC 1.3.1.20) activity in the cytosol of guinea-pig testis was separated into two major and two minor peaks by Q-Sepharose chromatography; one minor form was immunologically cross-reacted with hepatic aldehyde reductase. The two major enzyme forms were purified to homogeneity. One form, which had the highest amount in the tissue, was a monomeric protein with a molecular weight of 32,000 and isoelectric point of 4.2, showed strict specificity for benzene dihydrodiol and NADP+, and reduced pyridine aldehydes, glyceraldehyde and diacetyl at low rates. Another form, with a molecular weight of 36,000 and isoelectric point of 5.0, oxidized n-butanol, glycerol and sorbitol as well as benzene dihydrodiol in the presence of NADP+ or NAD+, and exhibited much higher reductase activity towards various aldehydes, aldoses and diacetyl. The pI 5.0 form was more sensitive to inhibition by sorbinil and p-chloromercuriphenyl sulfonate than the pI 4.2 form and was activated by sulfate ion. The two enzymes did not catalyze the oxidation of hydroxysteroids and xenobiotic alicyclic alcohols and were immunologically different from hepatic 17 beta-hydroxysteroid-dihydrodiol dehydrogenase. The results indicate that guinea-pig testis contains at least two dihydrodiol dehydrogenases distinct from the hepatic enzymes, one of which, the pI 5.0 enzyme form, may be identical to aldose reductase.     

Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.