Transaminations catalysed by brain glutamate decarboxylase.

In addition to normal decarboxylation of glutamate to 4-aminobutyrate, glutamate decarboxylase from pig brain was shown to catalyse decarboxylation-dependent transamination of L-glutamate and direct transamination of 4-aminobutyrate with pyridoxal 5'-phosphate to yield succinic semialdehyde and pyridoxamine 5'-phosphate in a 1:1 stoichiometric ratio. Both reactions result in conversion of holoenzyme into apoenzyme. With glutamate as substrate the rates of transamination differed markedly among the three forms of the enzyme (0.008, 0.012 and 0.029% of the rate of 4-aminobutyrate production by the alpha-, beta- and gamma-forms at pH 7.2) and accounted for the differences among the forms in rates of inactivation by glutamate and 4-aminobutyrate. Rates of transamination were maximal at about pH 8 and varied in parallel with the rate constants for inactivation from pH 6.5 to 8.0. Rates of transamination of glutamate and 4-aminobutyrate were similar, suggesting that the decarboxylation step is not entirely rate-limiting in the normal mechanism. The transamination was reversible, and apoenzyme could be reconstituted to holoenzyme by reverse transamination with succinic semialdehyde and pyridoxamine 5'-phosphate. As a major route of apoenzyme formation, the transamination reaction appears to be physiologically significant and could account for the high proportion of apoenzyme in brain.     

Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.     

Reactivation of substrate-inactivated brain glutamate decarboxylase

The effects of ATP and inorganic phosphate (Pi) on the reactivation of glutamate apodecarboxylase by its cofactor pyridoxal-5'-phosphate (pyridoxal-P) was studied. Apoenzyme was prepared by preincubation with glutamate. Apoenzyme prepared with glutamate alone was reactivated slowly and incompletely by adding a saturating concentration of pyridoxal-P (20 microM). Reactivation was slightly enhanced by 1-10 mM Pi. Reactivation by pyridoxal-P plus Pi was greatly enhanced by the presence of low concentrations (less than 100 microM) of ATP during the preparation of apoenzyme with glutamate. Reactivation was much lower if Pi was omitted. Enhancement of reactivation by ATP was due to its effect during apoenzyme formation, since ATP did not enhance reactivation if added only during reactivation and since the enhancing effect persisted after the removal of free ATP by chromatography on Sephadex G-25 after apoenzyme preparation and before reactivation. Reactivation was inhibited by high concentrations of ATP (greater than 100 microM), possibly by competition of ATP for the cofactor binding site. Four factors (glutamate, pyridoxal-P, ATP, and Pi) control a cycle of inactivation and reactivation that appears to be important in the regulation of brain glutamate decarboxylase.     

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.     

Multiple forms of acetylcholinesterase from pig brain

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100-KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.     

Post-mortem degradation of brain glutamate decarboxylase

The post-mortem stability of the GABA synthesizing enzyme glutamate decarboxylase (GAD) was studied by using SDS–PAGE and quantitative immunoblotting to measure the rates of degradation of GAD in the cerebral cortex, hippocampus, and cerebellum of rats and mice as a function of time after death. The intact 65- and 67-kDa isoforms of GAD (GAD₆₅ and GAD₆₇) disappeared gradually over a 24-h period. In both rats and mice, the degraded GAD appeared as a band with an apparent molecular mass of 55–57 kDa; no significant amounts of smaller forms were observed. The 55–57 kDa band reacted with antiserum W887, which recognizes a shared epitope at the carboxyl-terminal end of both GADs, indicating that GAD was cleaved near the amino-terminal end of the molecule. GAD₆₇ was cleaved at a site between the amino-terminus and the epitope for antiserum W883 (located within residues 79–93 of GAD₆₇), as antiserum W883 stained a 56-kDa band on the blots. The appearance of degraded GAD paralleled the loss of total GAD (GAD₆₅+GAD₆₇), and after 24 h the 55–57 kDa band accounted for 97, 88, and 59% of the intact GAD lost from rat cerebellum, cerebral cortex and hippocampus. On a percentage basis, GAD₆₇ was degraded more rapidly than was GAD₆₅ in all brain regions studied. The loss of GAD activity was greater in rat than mouse brain, even though the percent loss of intact GAD protein was similar.     

Stereochemical action of mouse brain glutamate decarboxylase

4-[4-²H]Aminobutyrate was prepared by incubation in ²H₂O of glutamate with a partially purified glutamate decarboxylase from mouse brain. The 4R configuration was assigned to the compound on the basis of ¹H nmr analysis of the ω-camphanoylamide of its methyl ester in the presence of Eu(dpm)₃. Moreover 4-[4(S)4-³H,U-¹⁴C]aminobutyrate was shown to be formed from [2(S)2-³H,U-¹⁴C]glutamate by the same enzyme fraction. It is therefore demonstrated that glutamate decarboxylation catalyzed by this enzyme preparation occurs with retention of configuration.     

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca²⁺/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.     

Characterization of three distinct forms of lipolytic enzyme in a commercialCandida lipase preparation

Summary Three distinct forms of lipolytic enzyme were identified in a commercialCandida lipase preparation. Two of these lipases (lipases A & C) were isolated and characterized. Lipase A had a higher optimal reaction pH and a better thermal stability than those of lipase C. Lipase A and C displayed different acyl chain length specificity on the lipolysis of p-nitrophenol esters.     

Characterization of a glutamate decarboxylase (GAD) gene from Lactobacillus zymae

Lactic acid bacteria (LAB) were isolated from Kimchi, a Korean traditional fermented vegetable food. LAB accumulating GABA (γ-aminobutyric acid) in the culture media were screened by TLC analysis. One isolate, GU240, produced the highest amount of GABA among the 3,000 isolates and identified as a Lactobacillus zymae strain. Glutamate decarboxylase (GAD) gene was cloned and over-expressed in E. coli BL21(DE3) using pET26b(+). The recombinant GAD was purified by using a Ni–NTA column. Its size was 53 kDa by SDS-PAGE. Maximum GAD activity was at pH 4.5 and 41 °C and the activity was dependent on pyridoxal 5′-phosphate. K_m and V_max of LzGAD were 1.7 mM and 0.01 mM/min, respectively, when glutamate was used as a substrate.     

Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain

Three types of protein kinase C, designated types I, II, and III, were purified from rat brain cytosol, and have been shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. Their relative activities in the whole brain tissue were roughly 26, 49, and 25% with H1 histone as a substrate. Type II enzyme was an unequal mixture of two subspecies (roughly 1:7) encoded by beta I and beta II sequences which differ from each other only in a short range of their carboxyl-terminal end regions. Although the three types have closely similar structures, they showed slightly different modes of activation and kinetic properties. Type I enzyme was less sensitive to diacylglycerol but was significantly activated by low concentrations of free arachidonic acid. Type II enzyme exhibited substantial activity without elevated Ca2+ levels, and responded well to diacylglycerol and, to some extent, arachidonic acid. The type III enzyme responded to diacylglycerol as well as to arachidonic acid. The mode of activation of the enzyme by arachidonic acid required elevated levels of Ca2+ but not phospholipid. In the presence of phospholipid, phorbol esters could activate all three types in a manner similar to diacylglycerol. Among various phospholipids tested, phosphatidylserine was the most effective for all three types. Type III enzyme was most sensitive to 1-stearoyl-2-arachidonylglycerol for activation. Conversely, type I enzyme was activated most efficiently by synthetic permeable diacylglycerols, such as 1,2-didecanoylglycerol and 1,2-dioctanoylglycerol. Many heavy metal ions exerted variable and distinct effects on the catalytic activities of these three types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.     

Characterization of three distinct flavonol O-methyltransferases from Chrysosplenium americanum

The partially purified O-methyltransferase (OMT) system of Chrysosplenium americanum was found to catalyse the stepwise O-methylation of quercetin to its mono-, di- and trimethyl derivatives. It also utilized the partially methylated flavonol intermediates to form the next higher order of O-methylated products; thus indicating the involvement of several OMTs. The latter were resolved by chromatofocusing into three distinct peaks of enzyme activity which focused at pI values 4.8, 5.4 and 5.7. The former enzyme O-methylated quercetin at the 3-position, whereas the latter two O-methylated 3, 7-di-O-methyl quercetagetin at the 3′- and 6-positions, respectively. None of the focused enzymes accepted caffeic acid, or other flavonoids such as kaempferol or luteolin, as substrates; thus indicating specificity towards flavonols with 3′, 4′- substitution. The three OMTs had similar MWs and the K_m values for their substrates were of the same order of magnitude. The biochemical role of these novel enzymes is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.     

Characterization of three distinct catalytic forms of human tryptase-beta: their interrelationships and relevance

Human tryptase-beta (HTbeta) is a serine protease that is isolated as a tetramer of four identical, catalytically active subunits (HTbeta-AT). Tetramer activity is not affected by protein-based physiological inhibitors but instead may be regulated by an autoinactivation process we have called spontaneous inactivation. Unless stabilized by heparin or high salt, the active tetramer converts to an inactive state consisting of an inactive-destabilized tetramer that reversibly dissociates to inactive monomers upon dilution. We refer to this mixture of inactive species as siHTbeta and show in this study that previous reports of monomeric catalytic forms are derived from this mixture. siHTbeta itself did not hydrolyze model substrates but unlike the tetramer did react slowly with the serpin alpha2-antiplasmin (alpha2-AP), suggesting a highly limited catalytic potential. In the presence of heparin (or other highly charged polysaccharides), we demonstrate that siHTbeta formed a well-defined complex with the heparin (siHTbeta-HC) that reacted 70-fold faster with alpha2-AP than siHTbeta and also hydrolyzed model substrates and fibrinogen. Formation of siHTbeta-HC was limited to dilute subunit solutions since high subunit concentrations resulted in the reformation of the active tetramer. By compensating for changes in the strength of heparin binding, siHTbeta-HC could be formed over the pH range of 6.0-8.5. The activity dependence on pH was bell-shaped with highest activity between pH 6.8 and pH 7.5. In contrast, HTbeta-AT activity showed no dependence upon heparin, increased over the pH range of 6.0-8.5, and was much higher than that of siHTbeta-HC especially above pH 6.8. HTbeta-AT incubated with excess heparin of different size (3-15 kDa) was functionally stable at 25 degrees C but lost activity regardless of heparin size at 37 degrees C above pH 6.8. The change in stability, which is likely due to weakened heparin binding, did not result in the formation of a stable catalytic monomer. These results confirm that siHTbeta is for the most part an inactive species and that any active monomer is a consequence of heparin binding to siHTbeta under dilute conditions where unfavorable thermodynamics and/or kinetics restrict formation of active tetramer. Heparin binding under these conditions drives a limited reorganization of the active site to a conformation that is catalytic but not the equivalent of a subunit within the active tetramer.     

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.     

Differences in some properties of newborn and adult brain glutamate decarboxylase.

Some properties of glutamate decarboxylase (EC 4.1.1.15) activity in brain of newborn and adult mouse were studied comparatively. It was found that glutamate decarboxylase of the newborn brain was strongly inactivated by homogenization in hypotonic medium, centrifugation of isotonic sucrose homogenates, preincubation at 37 degrees C or the addition of Triton-X-100, whereas the adult brain enzyme was practically unaffected by any of these conditions. It was also found that the newborn glutamate decarboxylase was less activated by pyridoxal 5'-phosphate and less inhibited by pyridoxal 5'-phosphate oxime-O-acetic acid, than the adult enzyme. These differences do not exist for brain dihydroxyphenylalanine decarboxylase (EC 4.1.1.26) and are not due to the release of inhibitors from the newborn brain. On the basis of the results obtained it is postulated that two forms of glutamate decarboxylase exist in brain: a newborn form, which is unstable and has high affinity for pyridoxal 5'-phosphate, and an adult form, which is much more stable and has low affinity for pyridoxal 5'-phosphate. The possible implications of these findings in the establishment of the gamma-aminobutyric acid dependent synaptic inhibitory mechanisms during development are discussed.