Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue

The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B(6)) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K(m) of 8.7 microM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes.     

Role of Amino Acid Residues on the GS Region of Stichopus Arginine Kinase and Danio Creatine Kinase

Stichopus arginine kinase (AK) is a unique enzyme in that it evolved not from the AK gene but from the creatine kinase (CK) gene: the entire amino acid sequence is homologous with other CKs apart from the guanidine specificity region (GS region), which is identical in structure to that of AK. Ten independent mutations were introduced around the GS region in Stichopus AK. When an insertion or deletion was introduced near the GS region, the Vmax of the mutant enzyme was dramatically decreased to less than 0.1% of the wild type, suggesting that the length of the GS region is crucial for the recognition of the guanidine substrate. Replacement of Phe63 and Leu65 to Gly in the Stichopus enzyme caused a remarkable increase in the Kmarg. This indicates that Phe63 and Leu65 are associated with the arginine substrate-binding affinity. The hydrogen bond formed between the Asp62 and Arg193 residues is thought to play a key role in stabilizing the closed substrate-bound structure of AK. Mutants that eliminated this hydrogen bond had a considerably decreased Vmax, accompanied by a threefold increase in Kmarg. It is noted that the value of the Kmarg of the mutants became very close to the Kdarg value of the wild type. Six independent mutations were introduced in the GS region of Danio M-CK. Almost equivalent values of Kmcr and Kdcr in all of the mutants indicated that a typical synergism was completely lost. The results suggested that the Ile69 to Gly mutant, displaying a high Kmcr and a low Vmax, plays an important role in creatine-binding. This is consistent with the observation that in the structure of Torpedo CK, Ile69 provides a hydrophobic pocket to optimize creatine-binding.     

Two Forms of Rat Brain Glutamic Acid Decarboxylase Differ in Their Dependence on Free Pyridoxal Phosphate

There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal-5'-phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45 degrees C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonic, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl-trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of gamma-amino-butyric acid (GABA) level is also discussed.     

A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization,Gene Cloning and Primary Structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

家蚕磷酸吡哆醛激酶cDNA的克隆和酶活表征

吡哆醛激酶 (EC 2.7.1.35) 在 ATP 和 Zn2 的存在下,催化吡哆醛的磷酸化反应生成磷酸吡哆醛 (PLP)。在生物体内许多酶促反应中,PLP 是一种重要的辅酶因子。家蚕和哺乳动物一样,需依赖食物中的维生素 B6前体来合成 PLP。文章描述了利用家蚕基因组数据库序列信息及使用 PCR 方法,克隆出编码家蚕吡哆醛激酶的 cDNA (GenBank 登录号:DQ452397)。克隆到的 cDNA 含有一个 894 bp 的完整可读框,编码一条分子量为 33.1 kDa,含 298 个氨基酸残基的蛋白质。序列比对显示此蛋白质序列与人类吡哆醛激酶蛋白序列具有 48.6%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但其氨基酸残基数比哺乳动物和植物克隆到的吡哆醛激酶残基数均少 10 多个残基。多序列比对结果显示,吡哆醛激酶中几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换为其他种类氨基酸残基。采用 T7 启动子和 T7 聚合酶表达系统对克隆到的 cDNA 进行了原核表达并对表达粗提产物进行了酶活检测。实验结果显示表达得到的可溶性蛋白产物占其总蛋白量为 10%,细胞粗提物具有活力为 30 nmol/min/mg 的吡哆醛激酶活性,结果证实了克隆到的 cDNA 编码家蚕中的吡哆醛激酶。     

Purification of a Phosphatidylinositol 4-Phosphate Kinase from Bovine Brain Membranes

A phosphatidylinositol 4-phosphate (PIP) kinase (EC 2.7.1.68) was purified from bovine brain membranes in a six-step procedure involving solubilization of the enzyme with 170 mM NaCl followed by chromatography on diethylaminoethyl-cellulose, phosphocellulose, Ultrogel AcA44, hydroxylapatite, and ATP-agarose. The enzyme preparation was nearly homogeneous and was purified 5,600-fold with a final specific activity of 85 nmol/min/mg of protein and a yield of 20%. Its molecular mass was 110 kilodaltons, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific for PIP; phosphorylation of phosphatidylinositol and diacylglycerol was not observed.     

High-Pressure Extraction of Membrane-Associated Protein Kinase C from Rat Brain

Extraction of rat brain membrane-associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain-associated activity was extracted from membranes. The pressure-extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure-extracted activity. The enzyme is extracted at greater than 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane-associated protein kinase C-lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Identification and Characterization of Protein Kinase FA/ Glycogen Synthase Kinase 3 in Clathrin-Coated Brain Vesicles

Abstract: Mg-ATP-dependent protein phosphatase activating factor [kinase F_A/glycogen synthase kinase 3 (GSK-3)] has been identified in highly purified clathrin-coated vesicles (CCVs) isolated from pig brain. Kinase F_A was found to exist in an inactive state but can be activated by 1% Triton X-100 or [ M /Tris-HC] extraction in brain CCVs. Activation of kinase F_A in CCVs is due to disassociation of the kinase from CCVs as demonstrated on sucrose density-gradient ultracentrifugation and Sepharose CL-4B gel filtration. Using purified brain CCVs as substrates, kinase F_A enhanced the endogenous phosphorylation of assembly protein complexes in the molecular weight range of 100,000-130,000 severalfold, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Comparisons with well-defined brain CCV-associated endogenous protein kinases such as pp50 kinase/AP50 and casein kinase 2 provide evidence that kinase F_A/GSK-3 represents a third potent and unique CCV-associated protein kinase distinctly different from the previously described CCV protein kinases, suggesting the possible involvement of kinase FA in the regulation of CCV functions in the brain. The results also support the notion that protein kinase F_A is involved in cell surface signal transduction in the CNS.     

Glutamate-Dependent Active-Site Labeling of Brain Glutamate Decarboxylase

A major regulatory feature of brain glutamate decarboxylase (GAD) is a cyclic reaction that controls the relative amounts of holoenzyme and apoenzyme [active and inactive GAD with and without bound pyridoxal 5'-phosphate (pyridoxal-P, the cofactor), respectively]. Previous studies have indicated that progression of the enzyme around the cycle should be stimulated strongly by the substrate, glutamate. To test this prediction, the effect of glutamate on the incorporation of pyridoxal-P into rat-brain GAD was studied by incubating GAD with [32P]pyridoxal-P, followed by reduction with NaBH4 to link irreversibly the cofactor to the enzyme. Adding glutamate to the reaction mixture strongly stimulated labeling of GAD, as expected. 4-Deoxypyridoxine 5'-phosphate (deoxypyridoxine-P), a close structural analogue of pyridoxal-P, was a competitive inhibitor of the activation of glutamate apodecarboxylase by pyridoxal-P (Ki = 0.27 microM) and strongly inhibited glutamate-dependent labeling of GAD. Analysis of labeled GAD by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed two labeled proteins with apparent molecular masses of 59 and 63 kDa. Both proteins could be purified by immunoaffinity chromatography on a column prepared with a monoclonal antibody to GAD, and both were labeled in a glutamate-dependent, deoxypyridoxine-P-sensitive manner, indicating that both were GAD. Three peaks of GAD activity (termed peaks I, II, and III) were separated by chromatography on phenyl-Sepharose, labeled with [32P]pyridoxal-P, purified by immunoaffinity chromatography, and analyzed by SDS-polyacrylamide gel electrophoresis. Peak I contained only the 59-kDa labeled protein. Peaks II and III contained the both the 59- and 63-kDa proteins, but in differing proportions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

草菇MAPKKK同源基因的克隆及序列分析

根据担子菌丝裂原活化蛋白质激酶激酶激酶(MAPKKK)蛋白的保守序列设计两对简并引物,通过巢式简并PCR方法获得草菇VV-MAPKKK基因中的保守片段,然后通过和草菇基因组信息比对,获得了VV-MAPKKK基因全长序列。VV-MAPKKK基因长度为4434bp,包含4个内含子,编码1405个氨基酸残基,推定的氨基酸序列与新型隐球菌(Cryptococcus neoformans)、巴西芽生菌(Paracoccidioides brasiliensis)和异旋孢腔菌(Cochliobolus heterostrophus)的MAPKKK同源蛋白相似性分别为58%、57%和56%。对VV-MAPKKK蛋白的系统发生学分析的结果表明,VV-MAPKKK与担子菌中的Hog信号传导途径的MAPKKK同源蛋白聚在同一进化支上,这些数据都支持所获得的VV-MAPKKK为Hog-MAPKKK蛋白在草菇中的同源物的推定。     

Isolation and Partial Characterization of an 80,000-Dalton Protein Kinase from the Micro vessels of the Porcine Brain

A novel serine/threonine-specific protein kinase was isolated from the microvessels of porcine brains. The molecular mass of the protein is 80,000 daltons, as judged by gel electrophoresis under denaturing conditions, or 122,000 daltons, on high-resolution gel permeation chromatography in the native state. The activity of this enzyme is stimulated by various histones or polyamines, like spermine or spermidine, but not by any of the common second messengers. The amino-terminal sequence data show no homologies to any of the published kinases, but rather to a heat-shock protein of unknown function.     

Partial Purification of Nuclear Protein Kinase from Small Dense Nuclei of Mouse Brain and the Effect of Chronic Morphine Treatment

Abstract: To deduce whether or not the nuclear protein kinase activity may be responsible for the change in phosphorylation of chromatin proteins during morphine tolerance-dependence, the nuclear protein kinases from small dense nuclei of mouse brain have been partially purified by ammonium sulfate fractionation and phosphocellulose column chromatography. Two peaks of cyclic AMP-independent nuclear protein kinase activity are eluted from the phosphocellulose column by a linear NaCl gradient. During morphine tolerance-dependence, the specific activity of peak I, but not of peak 11, is increased significantly relative to placebo controls. This increase in nuclear protein kinase activity may partially account for the elevated chromatin protein phosphorylation in small dense nuclei of mouse brain seen during morphine tolerance-dependence.     

Pyridoxal thiosemicarbazonate monohydrate of dimethylthallium(III): X-ray structure and spectroscopic properties

The title compoud, [TlMe₂(HL)(H₂O)] (HL = monoanion of pyridoxal thiosemicarbazone), crystallizers in the triclinic space group , No. 2). The HL⁻anion coordinates to the thallium atom, in an unusual mode through the S atom (Tl-S = 2.832(1) Å), and also forms a weak bond with the metal atom of a neighbouring molecule to make an asymmetric bridge (Tl′…S = 3.190(1) Å). The acidic proton retained in the thiosemicarbazonato anion is located on the oxygen of the phenolic hydroxyl group. The water molecule is only 2.630(4) Å from the metal, suggesting a rather strong bond that contrasts with the long distance between the thallium and the phenolic oxygen (Tl…O(1)′ = 3.124(4) Å). If both strong and weak intermolecular interactions are taken in account, the metal has distorted octahedral coordination with the methyl groups in apical positions. The solid state IR spectrum and ¹H, ¹³C and ²⁰⁵Tl NMR spectra in DMSO solution are also discussed.     

Protein Kinase FA/Glycogen Synthase Kinase-3 Predominantly Phosphorylates the In Vivo Site Thr97-Pro in Brain Myelin Basic Protein: Evidence for Thr-Pro and Ser-Arg-X-X-Ser as Consensus Sequence Motifs

Abstract: In a previous study, protein kinase F_A/glycogen synthase kinase-3 ( F_A/GSK-3 ) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase F_A/GSk-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F_A/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr⁹⁷-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase F_A/GSK-3, implicating a physiologically relevant role of F_A/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT⁹⁴(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [³²P]MBP phosphorylated by kinase F_A/GSK-3, further indicating that kinase F_A/GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin.     

Proline Reduces Creatine Kinase Activity in the Brain Cortex of Rats

Type II hyperprolinemia is an inherited disorder caused by a deficiency of ¹-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.     

Effect of Brain Ischemia on Protein Kinase C

We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6-min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane-bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane-bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca(2+)-dependent, antiprotease-insensitive down-regulation of PKC during incubation. This down-regulation was significantly enhanced by prior phorbol (PDBu) treatment.(ABSTRACT TRUNCATED AT 250 WORDS)