Histochemical Demonstration of Succinic Dehydrogenase in Ascites Tumor Cells

Fresh undiluted tumor ascites (0.05 ml) withdrawn from peritoneal cavity was placed immediately in a centrifuge tube containing 2.0 ml of an aqueous mixture prepared with 1 part each of the following solutions: 1% neotetrazolium chloride, 0.2 M sodium succinate and 0.1 M phosphate buffer, pH 7.4. The tube was incubated for 2 hr at 37°C and centrifuged for 3 min at 700 rev/min. The precipitate was washed with 0.85% saline solution and subsequently fixed with neutral 10% formalin for 10 min. After centrifugation, smears or squash preparations of the precipitate were prepared. Succinic dehydrogenase activity was demonstrated very distinctly and uniformly by the granular deposition of a deep purple pigment intracellularly.     

Enzymatic Assay of d-Xylose Isomerase with d-Sorbitol Dehydrogenase

Previously a cyclic pathway for the partial oxidation of propionyl-CoA to pyruvate has been proposed. Enzymatic evidence for the presence of the key reactions involved in this pathway is described and discussed herein. The condensation of propionyl-CoA with oxaloacetate into methylcitrate is shown to be catalyzed by an enzyme contained in cell-free extracts of Candida lipolytica; the enzyme seems to differ from the usual citrate synthase. Methylcitrate is easily convertible to a mixture of C₇-acids by the action of cell-free extract of the mutant strain. On the other hand, a similar mixture is changed into pyruvate and succinate by the action of cell-free extract of the parent strain. Evidence is given that methylisocitrate, one of the products of the conversion, is mainly cleaved by the action of an additional enzyme other than the usual isocitrate lyase. The accumulation of methylisocitrate in a large amount from odd-carbon n-alkanes by the mutant strain can be safely ascribed to the absence or a low level of this enzyme in the mutant strain.     

A New Enzymatic Microdetermination Procedure for Ethanol with Particulate Alcohol Dehydrogenase from Acetic Acid Bacteria

A new enzymatic method for microdetermination of ethanol has been established with particulate alcohol dehydrogenase from acetic acid bacteria and applied to the practical purposes. The enzyme had an optimum pH for ethanol oxidation at a fairly acidic region. Trace amounts of ethanol could be assayed by measuring the initial reaction rate as successful as by reading the end point of the reaction. Some advantages in using this enzyme for ethanol determination were pointed out comparing with NAD-linked alcohol dehydrogenase from yeast or horse liver. Impurity in the enzyme preparations, stability of reagents and coexistence of other substances in the assay mixture were not as critical as in NAD-linked enzyme. Acidic samples could also be directly determined for ethanol without preadjustment of sample pH.     

Direct Enzymatic Assay for Alcohol Oxidase, Alcohol Dehydrogenase, and Formaldehyde Dehydrogenase in Colonies of Hansenula polymorpha

A procedure is described for the qualitative direct identification of alcohol oxidase, alcohol dehydrogenase, and formaldehyde dehydrogenase in yeast colonies. The method has been applied successfully to isolate mutants of Hansenula polymorpha with altered glucose repression of alcohol oxidase.     

Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues

Abstract: We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.     

利用钙调素对乳酸脱氢酶活性影响定量测定钙调素

钙调素（calmodulin,CaM）在Ca2＋存在下能激活多种依赖CaM的靶酶．本研究对钙调素激活乳酸脱氢酶（lactatedehydrogenase,LDH．EC1．1．1．27）活性进行了探讨,其激活性质为非竞争性激活,并据此设计一种简便测定CaM的方法．1材料和方法1．1动物与制剂心肌和脑组织取自新生一周雄性小牛,NAD＋（上海酵母综合厂）,乳酸钠（北京化工厂）,DEAE－Cellulose、QAE－CelluloseA－50（上海化学试剂采购供应站）,NADH、氯丙嗪（chlorpromazine,CPZ）（Sigma）．1．2LDH的提取参照张龙翔［1］法略修改,将牛心肌粗提取液经DE…     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

A Pre-embedding Reaction Method for Localizing NADH Reductase and Succinic Dehydrogenase in Skeletal Muscle

Paraffin wax embedding methods suitable for demonstrating the distribution of enzyme activity in tissues sections are uncommon; most procedures rely on the use of frozen section techniques. This paper describes a system for demonstrating certain enzymes which involves incubation of the tissue with appropriate substrates before a Paramat wax embedding procedure. While it has distinct merits of its own, the procedure is eminently suitable for use where a cryostat is not available; it can also be readily applied to other enzymes and tissues.     

胰蛋白酶活性的定量测定方法

对甲苯磺酰基精氨酸甲酯(TAME)是胰蛋白酶的专一性底物. TAME经胰蛋白酶水解释放出的对甲苯磺酰基精氨酸与活性测定混合物中的NaOH反应, 导致溶液pH值的下降. 以酚红为指示剂, 通过测定555nm处光吸收值的降低可以监测pH的变化. 在0.001～0.3μg的范围内, 胰蛋白酶含量与555nm处光吸收值的降低呈线性关系.     

A Simple Assay Procedure for Mixtures of Hematoxylin and Hematein

A simple and rapid method for the simultaneous quantitative analysis of mixtures of hematoxylin and hematein uses the molar extinction coefficients of the pure substances calculated by Lalor and Martin (1959). Absorbance measurements of the samples dissolved in methanol are made at wavelengths of 292 nm and 445 nm, the wavelengths of maximum absorption of hematoxylin and hematein respectively. The hematoxylin absorbance at 292 nm is corrected for the presence of hematein.

Using this method it was found that of 12 commercial samples labelled “hematoxylin” all contained at least 90% of the compound. Hematein contents of these samples fell in the range 0.1% to 6.8%. In 9 commercial samples labelled “hematein” the hematein contents fell in the range 1.2% to 90.7%. The hematoxylin contents of these samples fell in the range of 1.0% to 82.7%.

This paper describes also a chromatographic method for the identification of hematein and its oxidation products.     

A Quantitative Procedure for Estimating Cotton Fiber Growth

An improved procedure for quantitation of cotton fiber development, the “stain-destain” method, is reported. Toluidine blue 0 was used to selectively stain fibers subsequently destained in an acid-alcohol solution. Absorbance of the dyecontaining destaining solution was used as a measure of fiber development, and expressed in terms of total fiber units (TFU), one OD unit at 624 nm having been assigned the value of one TFU. Optimum conditions for the procedure, including staining and destaining times and solution to ovule ratios were determined: (1) 20 ovules with associated fibers stained for 15 sec in 80 ml 0.018% toluidine blue O, (2) nonabsorbed dye removed by 60 sec wash, (3) ovuls destajned in 100 ml glacial acetic acid-ethanol-water (10:95:5), (4) absorbance determined after one hr destaining. The procedure is deemed accurate and precise for the purpose intended—quadtation of fiber development as modified by phytohormones or other treatments. Data are shown correlating TFU with fiber length through 14 days postanthesis and an example is given in which the method was used to determine the effect of combined application of gibberellic acid and indoleacetic acid on in vitro cotton fiber development.     

A Modified Soxhlet Extractor for Histologics and Histochemical Use

If the standard Soxhlet continuous-extraction apparatus is modified by the addition of a water jacket around thextraction chamber, the temperature of tissue undergoing extraction can be kept safely below damaging limits. Subsequent routine processing and examination of tissues after the cool extraction showed that they were less friable and had fewer structural distortions than those extracted with the uncooled, standard type c apparatus.     

Malate: A Possible Source of Error in the NAD Glutamate Dehydrogenase Assay

The effects of externally induced metabolic perturbations areoften studied through changes of the enzyme activity patternsin crude plant extracts. From glutamate dehydrogenase (GDH)it is reported that environmental changes not only influencethe amount of the enzymatic activity, but also the ratio ofthe aminating to the deaminating activities (NADH/NAD⁺ ratio).Using crude cell extracts of suspension cultures of wheat (Triticumaestivum L. cv. Heines Koga II) we find evidence that the pretreatmentof the homogenate directly influences this ratio. Dialysis ofthese crude cell extracts resulted in a 70% loss of the NAD⁺activity, while the NADH activity remained unchanged. The deaminatingactivity in the dialysed extract could be completely restoredupon addition of a dialysable factor which was identified tobe malate. The interference of malate with the glutamate dehydrogenasereaction is caused through the action of malate dehydrogenaseand glutamate oxaloacetate transaminase which are both presentin high activities in the extracts. Only in exhaustively dialysedcell extracts can the proper deaminating GDH activity be determined.The results are discussed in the light of the controversialreports on the variable ratio of the NADH/NAD⁺ activity of GDH. Key words: Glutamate dehydrogenase, malate, NADH/NAD⁺, activity, Triticum aestivum