NAD-dependent Alcohol Dehydrogenase and NADP-dependent Alcohol Dehydrogenase from Strawberry Seeds

Strawberry seeds are shown to contain at least two alcohol dehydrogenases; one is NAD specific and reacts with ethanol and allyl alcohol, and the other is NADP specific and reacts with benzyl alcohol and geraniol. These two alcohol dehydrogenases were distinguished on disc electrophoresis. Their properties were different each other in ammonium sulfate fractionation, optimum reaction pH and thermostability.     

Purification and Properties of Methanol Dehydrogenase and Aldehyde Dehydrogenase from Methylobacillus glycogenes

Methanol dehydrogenase (MDH) and aldehyde dehydrogenase (ALDH) were purified to a homogenous state from Methylobacillus glycogenes, an obligate methylotroph. MDH (M_r 140,000) was composed of two different subunits (M_r 60,000 and 9,000) forming an α₂β₂ structure. MDH was indicated as a metalloquinoprotein containing one atom of calcium (Ca) per enzyme molecule. Binding of Ca was so tight that it was hard to remove Ca completely without denaturation of enzyme protein. A partially resolved enzyme resumed its original enzyme activity upon exogenous addition of Ca. Purified ALDH (M_r 144,000) was composed of two identical subunits of molecular mass of 72,000. ALDH was proved to be a quinoprotein in which PQQ is bound covalently.     

Thrombin-Facilitated Efflux of d-[3H]-Aspartate from Cultured Astrocytes and Neurons Under Hyponatremia and Chemical Ischemia

Thrombin effect increasing swelling-induced glutamate efflux was examined in cultured cortical astrocytes, cerebellar granule neurons (CGN), hippocampal and cortical neurons. Hypotonic glutamate efflux (monitored by d-[³H]aspartate) from cortical astrocytes was increased by thrombin (5 U/mL) to reach 16 % of the cell pool in 5 min. Thrombin had lower effects in CGN, and marginal effects in hippocampal and cortical neurons. These differences were related to the magnitude of thrombin-evoked cytosolic calcium rise. The protease-activated receptor 1 is expressed in astrocytes and neurons. In astrocytes exposed to chemical ischemia (sodium iodoacetate plus sodium azide) d-[³H]aspartate release showed a first phase (20–40 min) of initial low efflux which progressively increases; and a second phase (40–60 min) of larger efflux coincident with cell swelling. Efflux at the first phase was 52 % inhibited by the glutamate transporter blocker DL-threo-β-benzyloxyaspartic-acid (TBOA) and 11 % by the volume-sensitive pathway blocker phloretin. At the second phase, efflux was reduced 52 and 38 % respectively, by these blockers. In CGN d-[³H]aspartate efflux increased sharply and then decreased. This efflux was 32 % reduced by calcium omission, 46 % by TBOA and 32 % by 4-[(2-butyl-6,7dichloro-2-cyclopentyl-2,3-dihydro-1oxo-1H-inden-5-yl)oxy] butanoic-acid. Thrombin enhanced this release by 32 %. Ischemic treatment increased astrocyte mortality from 4 % in controls to 39 and 61 % in ischemia and ischemia plus thrombin, respectively. Cell death was prevented by phloretin. CGN viability was unaffected by the treatment. These results suggest that coincidence of swelling and thrombin during ischemia elevates extracellular glutamate prominently from astrocyte efflux, which may endanger neurons in vivo.     

<Emphasis Type="SmallCaps">l</Emphasis>-Aspartate dehydrogenase: features and applications

l-Amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of l-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member l-aspartate dehydrogenase (l-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of l-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type l-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few l-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes.     

Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.Mannitol has been described as one of the main compatible solutes in fungi (20) and may play a role as a storage carbon source (3) or a protectant against a variety of stresses (10, 16, 20, 22). Mannitol metabolism in fungi has been the subject of study for decades. It was proposed to exist in the form of a cyclic pathway, the mannitol cycle (9). This cycle consists of four steps enabling the conversion of fructose into mannitol and back to fructose (Fig. 1). The main role proposed for this cycle was regenerating NADPH (9, 10). Subsequently, many studies have questioned the existence of a mannitol cycle (reviewed in reference 20), and it has been shown that a mannitol cycle is not involved in NADPH regeneration in Stagonospora nodorum (19), Aspergillus niger (16), and Alternaria alternata (21). However, all enzymes of the cycle were detected in both sporulating and nonsporulating mycelia in A. niger (16), suggesting that a cycle could operate in this fungus. Fungi are able to use mannitol as a sole carbon source but do so in various ways (7).Open in a separate windowFig. 1.Putative mannitol cycle in fungi as proposed by Hult and Gatenbeck (9). HXK, hexokinase (EC 2.7.1.1); MTD, mannitol dehydrogenase (EC 1.1.1.138); MPD, mannitol-1-phosphate dehydrogenase (EC 1.1.1.17); MPP, mannitol-1-phosphate phosphatase (EC 3.1.3.22).d-Mannitol plays an important role in germination of Aspergillus conidia. In A. niger (23) and Aspergillus oryzae (8), mannitol accumulates in conidiospores and is utilized during the initial stages of germination. Production of mannitol appears to be largely dependent on mannitol-1-phosphate dehydrogenase (MPD) while mannitol dehydrogenase (MTD) contributes to a lesser extent (16, 19, 20).In this study we demonstrate that MTD and MPD as well as the expression of the corresponding genes (mtdA and mpdA) are spatially separated in colonies of A. niger. This demonstrates that a mannitol cycle does not exist in this fungus and shows that spores express specific genes that are involved in germination.     

Characterization of Enterobacteria by Starch-Gel Electrophoresis of Glucose-6-Phosphate Dehydrogenase and Phosphogluconate Dehydrogenase

Specific activities and electrophoretic mobilities of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase were determined in 38 isolates of the family Enterobacteriaceae and in 10 isolates of the related Pasteurella. The deficiency of glucose-6-phosphate dehydrogenase in P. pestis was verified. Enzymes obtained from different strains of the same species exhibited an unexpected degree of heterogeneity. For example, 8 and 11 apparent variants of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase, respectively, were found in 14 strains of Escherichia coli. Although similar frequencies of heterogeneity were noted in 7 strains of P. pseudotuberculosis, 5 species of Shigella, and 8 species of Salmonella, differences in mobility were generally small in comparison with those observed between strains of E. coli. Values obtained for the pasteurellae, shigellae, and salmonellae, thus fell within narrow ranges that may prove typical for the genera. However, most of these ranges, as well as many values observed for single species of other genera, were overlapped by the wide range recorded for E. coli. The significance of this observation was discussed with respect to the relative age and taxonomic position of the organisms in question. The method could be used to distinguish between most wild-type strains of the same species and should thus facilitate investigations of genetic transfer and epidemiology.     

Distribution and Solubilization of Particulate Gluconate Dehydrogenase and Particulate 2-Ketogluconate Dehydrogenase in Acetic Acid Bacteria

The distribution of two particulate enzymes, gluconate dehydrogenase (GDH) and 2-ketogluconate dehydrogenase (2KGDH), was investigated with cell free extract through 26 strains of genus Acetobacter and genus Gluconobacter. GDH activity was found in the cell free extracts from all strains of genus Gluconobacter and two species of genus Acetobacter, A. aceti and A. aurantium. High activity of 2KGDH was also found in the pigment-producing strains of genus Gluconobacter.

Best solubilization of particulate enzymes was attained with the highest recovery when 10 mg of Triton X–100 and 30 mg of protein of particulate fractions in 1 ml of 0.01 m phosphate buffer, pH 6.0, are incubated for 9 hr at 5°C with continuous stirring.

By comparison of the total enzyme activity of particulate enzymes with that of NAD(P)-linked enzymes in the cell free extract, it was obvious that the formation of ketogluconates by particulate enzymes was much more predominant, roughly over 100 times higher, as that of NAD(P)-linked enzymes.     

Glucose-6-phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase of Candida tropicalis Partial Purification and Some Properties

Glucose-6-phosphate (G6P) dehydrogenase and 6-phosphogluconate (6PG) dehydrogenase were partially purified about 53-fold and 47-fold, respectively, from the cell-free extract of glucose-grown Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. AMP acted as the competitive inhibitor against G6P and NADP in the G6P dehydrogenase reaction. This inhibition was remarkable at low concentrations of NADP, increasing the sigmoidicity of the NADP-saturation curve. On the other hand, 6PG dehydrogenase was not affected by AMP. Fructose-1,6-bisphosphate (FDP) and phosphoenolpyruvate (PEP) inhibited slightly G6P dehydrogenase. 6PG dehydrogenase was also weakly inhibited by FDP. Apparent Km values of G6P dehydrogenase were calculated as 1.8 × 10^?4 m for G6P and 3.1 × 10^?5 m for NADP. Those of 6PG dehydrogenase were 9.4 × 10^?5 m for 6PG and 2.8 × 10^?5 m for NADP.     

胆碱脱氢酶与脂质体的重组

 用预先制备的脂质体与含Triton X-100的胆碱脱氢酶保温随后用Bio-beadsSM-2去除去垢剂而使酶重组到脂质体,Triton X-100的浓度,磷脂-蛋白比,Mg~(2+),pH等均影响重组,脂质体的大小在重组中不是关键因素,重组酶的活力比纯化酶增加70％,重组酶对PMS的Km,相转变温度及活化能均与线粒体内膜接近而不同于纯化酶。     

高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

N-Methyl-<Emphasis Type="SmallCaps">D</Emphasis>-Aspartate Receptor in Human Prostate Cancer

Expression of the N-methyl-D-aspartate receptor (NMDAr) and its involvement in cellular proliferation is well-known in tumors of neuronal tissue, such as glioma and neuroblastoma. We have investigated NMDAr expression in the normal, hyperplastic and neoplastic human prostate by immunohistochemistry. Low stromal NMDAr immunostaining was observed in 2 of 12 (17%) normal prostate specimens, but epithelial NMDAr staining was not seen. Of 18 benign prostatic hyperplasia (BPH) specimens, none had stromal NMDAr staining, but 2 had low and 1 had high epithelial NMDAr immunoreactivity. Moderate to high NMDAr immunostaining was observed in the stroma of 60 of 145 (41%) prostate cancer (PCa) specimens. Epithelial NMDAr staining was low in 26 (18%) and moderate to high in 36 (25%) of 145 PCa specimens. We have also examined the effects of the NMDAr antagonist memantine on the growth of ten human cancer cell lines: four prostate, two breast and four colon. The NMDAr antagonist memantine inhibited in-vitro growth of all ten cell lines, with half-maximal growth-inhibition at 5 to 20 μg/ml (23 to 92 μM) memantine. An NMDA agonist, L-cysteinesulfinic acid, stimulated cellular proliferation of all ten cell lines, with maximal growth-stimulation (30% to 75%, depending on the cell line) observed between doses of 33 to 66 μM. Our data provide evidence for the expression and activity of NMDAr in prostate cancer.     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

Immunological Difference between Glucose-6-P Dehydrogenase and Hexose-6-P Dehydrogenase from Human Liver

SHAW and Barto¹ have demonstrated the presence of an autosomally inherited glucose-6-P dehydrogenase (G6PD) in the deer mouse. Subsequently, Ohno et al.² found a similar enzyme in trout and showed that this enzyme and the autosomally inherited mouse enzyme differed from the sex-linked G6PD in possessing marked catalytic activity with galactose-6-P. This autosomally inherited G6PD was therefore named hexose-6-P dehydrogenase (H6PD)^2,3. It was shown to oxidize glucose-6-P, galactose-6-P, mannose-6-P and 2-deoxy glucose-6-P with a K_m of the order of 10^?5 M. It also oxidizes glucose with a K_m of 0.7 M³. It appears to be identical to the so-called “glucose dehydrogenase”. The enzyme utilizes both NAD and NADP and is microsome-bound. G6PD is localized in the soluble fraction of the cells of various tissues. Although it has been shown that two dehydrogenases from liver have different substrate specificity, molecular weight and elec-trophoretic mobility^3,4, it has been suggested that the two enzymes are merely isozymes and they might be interconvertible^5–7. We have now partially purified the two enzymes from human liver and show that they have different immunological properties.     

Alcohol Dehydrogenase of Apple

The alcohol dehydrogenase prepared from apple (Malus domesticaBorkh.) possesses both NADH and NADPH-linked activities, whenassayed with acetaldehyde as substrate. The pyridine nucleotidesbind to the same catalytic site on the enzyme. The alcohol dehydrogenasecan also catalyse the reduction of C₃–C₆ aldehydes witheither NADH or NADPH as cofactor.