甜菜碱醛脱氢酶在菠菜叶细胞中的定位

将菠菜叶片匀浆后．用差速离心和梯度率心分离叶绿体、过氧物酶体、微粒体等细胞器和１０００００×ｇ上清法部分。用酶活测定法测定各部分甜菜碱醛脱氢酶（ＢＡＤＨ）的活性;用免疫扩散法鉴定各组分的ＢＡＤＨ。除叶绿体外,过氧物酶体、微粒体．以及１０００００×ｇ上清液中也存在ＢＡＤＨ。     

The effects of temperature and chorionic gonadotropin on the functional properties of lactate dehydrogenase from oocytes of loach Misgurnus fossilis

Acclimation of loach to relatively low and high temperatures gives rise to changes in the properties of LDH from oocytes. The minimum of apparent K_m values for pyruvate of LDH from immature oocytes of fish acclimated to low temperature was registered at low assay temperatures, whereas adaptation to high temperatures leads to the enzyme showing a minimum at high temperatures. In thermal acclimation of fish the K_m minimum value drifts during 15 days. During oocyte maturation, the K_m minimum drifts during 40 hr. The differences in the kinetic features of LDH from oocytes both at thermal acclimation and during oocyte maturation are not associated with the appearance of new isozymes.     

Hydrogen Peroxide and the Proliferation of Bhk-21 Cells

Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H₂O₂, such as the superoxide dismutase mimic, copper II—(3,5-diisopropylsalicylate)₂, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 μM exogenous H₂O₂ to the culture medium. Under these conditions, however, intracellular levels of H₂O₂ are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H₂O₂ and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H₂O₂, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to “trigger” their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H₂O₂     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)     

Identification of two different glyceraldehyde-3-phosphate dehydrogenases (phosphorylating) in the photosynthetic protist Cyanophora paradoxa

Two different glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) activities, namely NAD- and NADP-dependent, have been found in cell extracts of the cyanelle-bearing photosynthetic protist Cyanophora paradoxa. Whereas the two G3P dehydrogenase activities were detected with similar specific activity levels (0.1 to 0.2 U/mg of protein) in extracts of the photosynthetic organelles (cyanelles), only the NAD-dependent activity was found in the cytosol. Thus, a differential intracellular localization occurred. The perfect overlapping of the two G3P dehydrogenase activity peaks of the cyanelle in both hydrophobic interaction chromatography and subsequent FPLC (fast protein liquid chromatography) gel filtration indicated that the two activities were due in fact to a single NAD(P)-dependent G3P dehydrogenase (EC 1.2.1.-) with a molecular mass of 148,000. SDS-PAGE of active fractions from FPLC gel filtration showed that the intensity of the major protein band (molecular mass, 38,000) of the enzyme preparation clearly paralleled the activity elution profile, thus suggesting a tetrameric structure for the cyanelle dehydrogenase. On the other hand, FPLC gel filtration analysis of the cytoplasmic fraction revealed a NAD-dependent G3P dehydrogenase with a native molecular mass of 142,000, being equivalent to the classical glycolytic enzyme (EC 1.2.1.12) present in the cytosol of all the organisms so far studied. The significance of these results is discussed taking into account that the cyanobacteria, photosynthetic prokaryotes which share many structural and biochemical features with cyanelles and are considered as their ancestors, have a similar NAD(P)-dependent G3P dehydrogenase.Abbreviation FPLC Fast protein liquid chromatography     

Alcohol dehydrogenase (EC 1.1.1.1) isozymes as markers at 2,4-dichlorophenoxyacetic acid × kinetin combinations in callus cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH; EC 1.1.1.1) isozymes were investigated in tissue ofCereus peruvianus cultured in different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Five ADH isozymes were detected in starch gel and showed different patterns in seeds, seedlings, calli cultured at 32 and 22°C, and plants regenerated from calli cultured in three 2,4-D and kinetin combinations. Four phenotypes formed by different combinations of ADH-2, ADH-3, ADH-4, and ADH-5 were detected in calli cultured at 32°C and in plants regenerated from calli. ADH-1 isozyme was detected only in calli subcultured for 1 or 2 weeks at 22°C and was indicated as a marker of stress conditions that affect the growth ofC. peruvianus callus tissues in culture. ADH phenotypes with either a higher or a lower number of isozymes were detected in different proportions in the callus tissues cultured in media containing different 2,4-D and kinetin ratios. ADH isozyme patterns were found to be sensitive markers at the highest kinetin concentration or at high kinetin/2,4-D ratios. The results indicate a high correlation between the ADH isozyme patterns and the capacity for regeneration. Thus, ADH isozymes are indicated as good biochemical markers and as a powerful tool for monitoring studies ofC. peruvianus callus cultures.This research was supported by the CNPq.     

Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

Substrate and inhibitor specificities of the thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster

Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK _m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee.     

WHEN MORPHOLOGY MISLEADS: INTERPOPULATION UNIFORMITY IN SEXUAL SELECTION MASKS GENETIC DIVERGENCE IN HARLEQUIN BEETLE-RIDING PSEUDOSCORPION POPULATIONS

Differences in secondary sexual characteristics of males often provide the most conspicuous means of distinguishing between closely related species. Does this therefore imply that the absence of differentiation in exaggerated male traits between allopatric populations provides evidence of a single, genetically cohesive species? We addressed this question with a comprehensive investigation of two populations (French Guiana and Panama) of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides. This highly sexually dimorphic pseudoscorpion is currently described as a single species, ranging throughout the Neotropics. Our morphometric analyses detected minimal differentiation between the two populations in all nine external morphological characters measured, including sexually dimorphic traits in males. Only in traits of the spermatophore was there any appreciable level of differentiation. Behavior differentiation and prezygotic reproductive isolation were also limited: 78.3% of males successfully transferred sperm to “foreign” females, and in 63.9% of these cases, females' eggs were successfully fertilized. By contrast, extensive divergence existed in two of nine electrophoretic loci, including an essentially fixed-allele difference at the Ldh locus. Most significantly, postzygotic reproductive isolation was complete, with heteropopulation zygotes invariably aborting early in development. These results strongly suggest that the two populations are, in fact, sibling species, a conclusion supported by our recently published findings on their marked divergence in minisatellite DNA. How can such interpopulation homogeneity in male sexually dimorphic traits exist in the face of strong genetic divergence? We propose that sexual selection, oscillating between favoring small and then large males, maintains such high levels of male variability within each population that it has obscured a speciation event in which genetic divergence and postzygotic incompatibility have clearly outpaced the evolution of prezygotic reproductive isolation.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.