Histidine decarboxylase from rat and rabbit brain: Partial purification and characterization

Histidine decarboxylase, the synthetic enzyme for histamine, was partially purified from regions of rat or rabbit brain rich in the enzyme. The enzyme was purified using ion exchange and hydrophobic column chromatography and chromatofocusing. Approximately 70-fold and 110-fold enrichments were attained from rat and rabbit brain, respectively. Rat and rabbit brain histidine decarboxylase had isoelectric points of pH 5.4 and 5.6, Km values of 80 M and 120 M histidine and Vmax values of 210 and 625 pmol histamine formed/hr-mg protein, respectively. The partially purified histidine decarboxylase from both sources was dependent on pyridoxal phosphate for maximal activity and was inhibited by -fluoromethylhistidine, nickel chloride and cobaltous chloride but was not inhibited by impromidine, -methyldopa, DTNB, zinc chloride or mercuric chloride. The enzyme had a broad pH optimum between pH 7.2 and 8.0. These studies provide further information on the characteristics of mammalian histidine decarboxylase from brain.     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.     

Histidine decarboxylase: Isolation and molecular characteristics

High levels of histidine decarboxylase activity were measured in rat basophilic leukemia cells grown in ascitic form in 4 week old WKY/N rats. The potent inhibition of this enzyme by brocresine and -methylhistidine but not by -methyl DOPA identified it as a specific histidine decarboxylase. Gel filtration and polyacrylamide gel electrophoresis revealed a molecular weight of 125,000 for the native enzyme, similar to that of fetal rat liver histidine decarboxylase. Using rat basophilic leukemia cells as starting material, histidine decarboxylase was purified extensively in a seven step procedure. Electrophoresis under denaturing conditions revealed that histidine decarboxylase is a dimeric protein consisting of two identical subunits with a molecular weight of 62,000. The results indicate that rat basophilic leukemia cells provide a new and rich source for the purification of histidine decarboxylase.     

Rat liver cysteine sulfinate decarboxylase: purification, new appraisal of the molecular weight and determination of catalytic properties.

Rat liver cystein sulfinate decarboxylase (L-cystein sulfinate carboxylase) was purified approximately 500-fold. By cellulose acetate and polyacrylamide gel electrophoresis or by analytical ultracentrifugation, the purified enzyme appears to be nearly homogeneous. The Stokes radius (3.4 nm) and sedimentation coefficient (6.5 S) were determined. The molecular weight, calculated and experimentally estimated is around 100 000 and the enzyme is constituted of two identical subunits whose molecular weights are 55 000. The role of pyridoxal phosphate as coenzyme was demonstrated and the requirement for free sulhydryl groups for activity was studied. The ability of native pure cysteine sulfinate decarboxylase to also decarboxylate cysteate was stressed: therefore, we concluded that in rat liver a single protein catalyzed both reactions, although only the decarboxylation of cysteine sulfinate is of physiological interest.     

Purification and properties of malonyl-CoA decarboxylase from rat liver mitochondria and its immunological comparison with the enzymes from rat brain, heart, and mammary gland.

Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH₄)₂SO₄ precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which K_m and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA.     

Polyamine biosynthesis and vitamin B6 deficiency Evidence for pyridoxal phosphate as coenzyme for S-adenosylmethionine decarboxylase

Extracts of liver from vitamin B₆-deficient rats had only 50% of the S-adenosylmethionine decarboxylase activity of extracts of liver from control rats when assayed with no exogenous pyridoxal phosphate. When pyridoxal phosphate was included in the reaction mixture, both extracts exhibited the same activity, indicating that pyridoxal phosphate is the coenzyme for S-adenosylmethionine decarboxylase. There was no similar decreased activity in extracts of brain from vitamin B₆-deficient rats.The activity of the pyridoxal phosphate-dependent enzyme, ornithine decarboxylase, was increased in extracts of liver from vitamin B₆-deficient rats: 1.6-fold when assayed with no pyridoxal phosphate and 4-fold when assayed with pyridoxal phosphate.The concentrations of putrescine and spermidine were decreased 50% in liver of vitamin B₆-deficient animals, but only putrescine was decreased in brain. Putreanine was barely detectable in liver of vitamin B₆-deficient animals, but was unchanged in brain.     

GABA-TRANSAMINASES OF HUMAN BRAIN AND PERIPHERAL TISSUES—KINETIC AND MOLECULAR PROPERTIES

Abstract— Kinetic experiments with 4-aminobutyrate-2-ketoglutarate transaminase (GABA-T), partially purified from human brain tissue, supported a Bi Bi Ping-Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated K _m values were 0.31 m m for γ-aminobutyrate, 0.16 m m for α-ketoglutarate, and 3.8 μ m for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA-T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA-T inhibitors.
GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors.     

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.     

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.     

Purification and some properties of L-glutamate decarboxylase from human brain

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000-fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N-terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 x 10(-3) M for glutamate and 0.13 x 10(-6) M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3-mercaptopropionic acid (Ki = 2.7 x 10(-6) M) while the Ki values for allylglycine and chloride ion are 1.8 x 10(-2) M and 2.2 x 10(-2) M, respectively.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.     

Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity.

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1'-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.     

Multiple forms of rat liver cysteinesulfinate decarboxylase

Cysteinesulfinate decarboxylase, purified from male rat livers and homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is resolved into five distinct enzyme species (isoforms) by gel isoelectric focusing. Since the isoforms are present in fresh liver homogenates and do not arise by proteolysis, the enzyme is apparently heterogeneous in vivo. Although female rat livers contain only 5% of the cysteinesulfinate decarboxylase activity of male livers, immunological and enzymatic studies indicate that the distribution of isoforms is similar in both sexes. Rat brain and kidney also contain multiple isoforms which are cross-reactive with polyclonal antibodies prepared to the liver enzyme. The enzyme exhibits a protomer Mr of 53,000, and the native enzyme is shown by cross-linking studies to be dimeric. Purified enzyme contains no carbohydrate or phosphate and does not bind excess pyridoxal 5'-phosphate. Two pools of enzyme activity are resolved preparatively by chromatofocusing chromatography and have been examined with respect to substrate and inhibitor specificity. Both pools are most active toward L-cysteinesulfinate and L-cysteinesulfonate. Aspartate, homocysteinesulfinate, homocysteinesulfonate, 2-amino-3-phosphonopropionate, and glutamate are decarboxylated at rates less than 1% of that observed with L-cysteinesulfinate; D-cysteinesulfinate is not decarboxylated but is an effective inhibitor. The enzyme isoforms cannot be distinguished on the basis of substrate affinity or specificity. The enzyme is irreversibly inactivated by the mechanism-based inhibitors beta-methylene-DL-aspartate and beta-ethylidene-DL-aspartate. beta-Ethylideneaspartate, in contrast to the beta-methylene derivative, does not inhibit aspartate aminotransferase, an enzyme also important in cysteinesulfinate metabolism. beta-Ethylidene aspartate or related beta-ethylidene compounds may be useful in selectively altering cysteinesulfinate metabolism in vivo.     

Regulation of ornithine decarboxylase synthesis. Effect of a nutritional deficiency of vitamin B6

In vitamin B₆ deficiency there is an increase in the activity of the pyridoxal phosphate dependent enzyme ornithine decarboxylase. In the rat liver: the apoenzyme and holoenzyme activity increased 1.6 and 4 fold respectively. Concomitantly, putrescine and spermidine concentrations were halved. The lack of correspondence between product concentration and enzymic activity suggests a control mechanism other than ornithine decarboxylase activity.     

ADENOSYLMETHIONINE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Adenosylmethionine decarboxylase from rat brain has been found to be similar to the same enzyme isolated from other rat tissues in regard to kinetic parameters, pH optimum, putrescine requirement, and subcellular location. Evidence is presented that pyridoxal phosphate is not the functional cofactor in enzymatic decarboxylation by the rat brain preparation. The capacity for spermidine synthesis in developing rat brain was determined by measurement of the activity of adenosylmethionine decarboxylase. The activity increased dramatically after 10 days of postnatal age. This increase occurred after the period of maximum nucleic acid synthesis, an observation which suggests that spermidine may have a role in the functional development of the brain.     

Arginine decarboxylase from Lathyrus sativus seedlings. Purification and properites.

Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5-7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977-fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C-gamma gel, DEAE-Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220,000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 degrees C respectively. The enzyme follows typical Michaelis-Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co-factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal-HCl suggests that pyridoxal 5'-phosphate is involved as a co-factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, L-canavanine, homologue L-homoarginine and other basic amino acids like L-lysine and L-ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.     

Activation by adenosine-5'-triphosphate of glutamic decarboxylase from a subcellular fraction of mouse brain.

Glutamate decarboxylase from a mouse brain P2 fraction undergoes a twofold activation in the presence of 0.5 mM ATP. No such stimulation by ATP occurs if the enzyme is assayed in the presence of excess pyridoxal phosphate as cofactor. The ATP-induced stimulation is almost completely eliminated if the enzyme is dialysed before its assay. [lambda-32P]ATP present during the enzyme measurement is converted to [32P]pyridoxal phosphate. These results demonstrate that the activation produced by ATP is the result of the generation of cofactor during the course of the assay. This phenomenon may be a reflection of a control mechanism of glutamate decarboxylase activity.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE,PYRIDOXAL 5-PHOSPHATE AND GLUTAMATE-PYRIDOXAL 5-PHOSPHATE SCHIFF BASE1

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.