Endogen und exogen ausgelöste Änderung des Isoenzymspektrums der NAD-spezifischen Glutamatdehydrogenase im Sproß von Pisum sativum

Summary The isoenzymes of NAD-specific glutamic dehydrogenase (GDH) of Pisum sativum, separated by polyacrylamide gel electrophoresis, constitute two patterns, each of which covers seven individual isoenzymes. One pattern (GDH-I) is found in the cotyledons and young shoots. The second one (GDH-II) occurs together with at least some GDH-I isoenzymes in pea roots. In the shoots of older pea plants GDH-II isoenzymes become visible in addition to the GDH-I pattern.Section of the cotyledons (but not of the roots) of young pea seedlings causes the formation of the complete GDH-II isoenzyme pattern in the shoots within a few hours. It has been verified that the cotyledons specifically suppress the formation of the GDH-II pattern in the young shoot. In older plants which no longer depend on the cotyledons this effect is maintained somewhat less obviously by the root system.In experiments with isolated shoot segments or shoot tips it has been shown that NH ₄ ⁺ reinforces the formation of the GDH-II whereas glucose shows the opposite effect.The formation of the GDH-II isoenzymes in the presence of NH ₄ ⁺ is accompanied by an increase of the specific activity of GDH. Simultaneously the ratio of aminating activity (anabolic reaction) to deaminating activity (catabolic reaction) changes in favor of the anabolic reaction.The results support the supposition that the GDH-I and GDH-II isoenzyme patterns correspond to different molecular forms of one enzyme, the GDH-II representing a form with predominantly anabolic function and the GDH-I a form which has merely metabolic or catabolic function.     

Isoenzymes of glutamate dehydrogenase in plants

Glutamate dehydrogenase of several different plants was resolved by polyacrylamide gel electrophoresis into separate molecular forms and the isoenzymic patterns detected by the tetrazolium technique were compared. The number of isoenzymes and their electrophoretic mobilities varied among the different plants studied. The isoenzymes were found to have the same coenzyme specificity and to localize in the mitochondrial fraction of the cell in all the plants examined. Electrophoretic heterogeneity in tissue homogenates was observed in some of the plants studied. The pattern of isoenzymes of mungbean hypocotyl was followed and shown to change during germination.     

Changes in isoenzymes in embryo and endosperm of normal and opaque-2 Zea mays during imbibition

Electrophoretic patterns of soluble proteins, peroxidase, esterase, alcohol dehydrogenase (ADH) and glutamic dehydrogenase (GDR) from embryos and endosperm of normal and opaque-2 maize were studied after different periods of imbibition. The soluble protein pattern from endosperm of normal and opaque-2 differed both qualitatively as well as quantitatively. The embryo protein patterns were identical. Multiple forms (isoenzymes) were found for all the enzymes studied. The enzyme patterns changed during imbibition. Peroxidase and GDH patterns from embryos of normal and opaque-2 showed considerable differences during imbibition. Esterase and ADH pattern from embryo and endosperm of normal and opaque-2 showed few differences.     

Isoenzyme Activities during the Early Stages of Allium Radicle Germination

As a continuation of a study of the molecular events surrounding the establishment of a mature root apex, changes in the pattern and specific activity of the isoenzymes of aminopeptidase (AmP), esterase (EST), glucose-6-phosphate de-hydrogenase (G6PD) and glutamic-oxaloacetic transaminase (GOT) were investigated during the early germinative growth of the Allium radicle tip. Polyacrylamide disc gel electrophoresis revealed a total of 9 EST fractions. A shift in pattern and reactivity characteristic of pre-emergence to those characteristic of post-emergence was demonstrated. G6PD electropherograms displayed 5 isoenzyme bands. Relative specific activity analyses indicated that this enzyme may be more important during the very early stages of germination. Only quantitative differences were noted in the 3 AmP isoenzymes detected. The progressive increase in AmP isoenzyme activity during the period of maximal protein body degradation suggested a role for these exopeptidases in the degradation of reserve proteins. The 3 isoenzyme bands of GOT exhibited only quantitative changes during germination. Parallel increases in the quantity of soluble protein and GOT specific activity suggest that this isoenzyme is involved in the onion's nitrogen metabolism during germination.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Effect of drought on proteins and isoenzymes in rice during germination

Two drought tolerant varieties TKM-1 and TKM-2 and two drought susceptible varieties Jaya and Improved Sabarmati of rice were studied for soluble protein pattern and isoenzymes of malate dehydrogenase, glutamate dehydrogenase, esterase and peroxidase during germination at different water stress. MDH, GDH and esterase patterns were not affected, but the soluble proteins were changed. Peroxidase isoenzyme pattern from drought tolerant and susceptible varieties showed characteristic differences. The intensity of bands with higher electrophoretic mobility decreased in Jaya and Improved Sabarmati while in TKM-1 and TKM-2 the intensity of these bands did not change much after 72 hr water stress. In shoots of Jaya and Improved Sabarmati, the activity of the peroxidase isoenzymes decreased more than in TKM-1 and TKM-2 shoots with increase in water stress.     

Multiple molecular forms of Acanthamoeba lactic dehydrogenase

1. Acanthamoeba lactic dehydrogenase (D-lactate specific, EC 1.1.1.28) has been studied through the use of polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (PAGIEF). 2. Data from PAGE showed the presence of only a single macromolecular form of the enzyme in trophozoites, but PAGIEF data demonstrated that at least three LDH species occur. 3. Molecular weight determinations indicated a tetrameric assembly state for the enzyme. 4. Multiple molecular forms of the enzyme (isoenzymes) are encoded either by genes at two loci or by two alleles at a single LDH locus.     

Variation in seed protein and isoenzyme patterns in Cucurbita cultivars

The genetic variability in the seed proteins and the enzyme alcohol dehydrogenase (ADH) in representative species of the genus Cucurbita was studied. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gel. A specific protein components and ADH isoenzymes were established in the polymorphic banding patterns which can be applied individually or in combination as potential biochemical markers for breeding purposes.     

Malate Dehydrogenase Isoenzymes in Cotton Leaves of Different Ages

Malate dehydrogenase isoenzymes from the blades of different aged leaves of the cotton plant have been investigated. The total extractable malate dehydrogenase activity varied widely between leaves of different ages and different locations on the plant. Malate dehydrogenase zymograms developed from the extracts which contained significantly different levels of enzyme activity appear to indicate the presence of different groups of malate dehydrogenase isoenzymes in leaves of different ages. However, under appropriate conditions of polyacrylamide gel electrophoresis, the same number of malate dehydrogenase isoenzymes with the same relative mobilities were detected in all the leaves studied. These findings are discussed in relation to reports that malate dehydrogenase isoenzymes change with plant development or that they have different roles in the plant.     

Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio

The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, α and β, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of α- and β-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of β-polypeptide and of the more cathodal isoenzymes, whereas α-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the α- and the catabolic reaction with the β-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions.     

Lactate dehydrogenase isoenzyme transitions during skeletal differentiation in the mouse embryo

(1) In the mouse embryo there are changes in lactate dehydrogenase activity and isoenzyme pattern during the differentiation of cartilage and bone. (2) The specific activity of lactate dehydrogenase rises during chondrogenesis and falls during osteogenesis. (3) Identical isoenzyme transitions occur in parallel in both tissues: undifferentiated limb bud mesenchyme contains isoenzymes 1-5 whereas in both the cartilaginous and bony portions of a long bone developing from the mesenchyme, there is a progressive shift towards a predominance of the 'anaerobic' isoenzymes 4 and 5.     

Anomalous behaviour of yeast isocitrate dehydrogenase during isoelectric focusing

Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ;isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.     

Wachstum und N-Stoffwechsel sowie Anderungen des Isoenzymspektrums der Glutamatdehydrogenase (GDH) in batchvermehrten, licht- und dunkelkultivierten Zellsuspensionen von Pisum sativum

Growth, N-metabolism and isoenzyme pattern of glutamate dehydrogenase in batch-cultures of Pisum sativum cells under light and dark conditions. Cell suspension cultures of Pisum sativum L. derived from root and shoot sections of seedlings have been prepared and cultured in defined nutrient medium. Both the cells and the media were analysed daily for the N-fractions and carbohydrates during the growth period. The data obtained indicate specific correlations between growth and nitrogen and carbohydrate metabolism. At the beginning of the growth cycle ammonia as compared to nitrate was favoured in uptake. An increased uptake of nitrate occurred at the end of the linear growth phase when carbohydrate in the media was depleted. The uptake of sucrose was rapid during the whole growth cycle, only in the range of the linear growth phase the uptake stagnated for 3 or 4 days. During increased biosynthesis of nitrogenous compounds at the beginning of the growth cycle up to seven isoenzymes of the glutamate dehydrogenase could be separated by polyacrylamide gel electrophoresis. The isoenzyme pattern changed during the stationary growth phase, especially when the carbohydrate content in the medium decreased. There is some evidence that the isoenzyme pattern is influenced by carbohydrate metabolism.     

Isolation and partial characterisation of α-amylase components evolved during early wheat germination

The development of α-amylase (EC 3.2.1.1) activity in wheat was followed during 4 days of germination. The enzyme was purified and separated by gel chromotography into two distinct entities (α-amylase I and α-amylase II), with different molecular weights and isoelectric points. α-Amylase I contained a much higher content of sugars than α-amylase II, which decreased as the germination proceeded. The time sequence analysis of the starch degradation pattern showed that on the 4th day of germination, 15% of the total activity was present in α-amylase I and the rest in a-amylase II. Similarly, differences in the relative rates of synthesis of their isoenzymes were observed. α-Amylase I was resolved on the 4th day of germination, only into 3 isoenzymes, whereas α-amylase II could separate into 4 isoenzymes. The enzyme activity was however maximal in the most electropositive isoenzyme in both the components.     

Role of differential expression of <Emphasis Type="Italic">sdh1-1</Emphasis> and <Emphasis Type="Italic">sdh1-2</Emphasis> genes in alteration of isoenzyme composition of succinate dehydrogenase in germinating maize seeds

A probable mechanism of alteration of the isoenzyme composition of succinate dehydrogenase (SDH) due to differential expression of genes encoding subunit A was considered. The alteration of SDH activity during maize seed germination was investigated, and its maximal activity on day 4-5 of germination was found. The alteration of the sdh1-1 and sdh1-2 gene expression level during maize seed germination was evaluated using the quantitative polymerase chain reaction method. The presence of four forms of the studied enzymes, providing multiple SDH functions was found in maize inflorescence using electrophoresis in polyacrylamide gel.     

In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida

Summary Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms.     

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA : II. NAD- and NADP-Dependent Glutamic Dehydrogenases and Nicotinamide Adenine Dinucleotidase

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged.     

Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

The pattern of change in the activity of alcohol dehydrogenase in maize (Zea mays L.) scutellum during seed germination is not altered by 10 μg/ml cycloheximide or 50 μg/ml actinomycin D. The enzyme does not become density labeled when maize seeds are germinated in the presence of D₂O and ¹⁵NH₄Cl, indicating that no new alcohol dehydrogenase molecules are synthesized after the onset of germination. However, the activity of an endogenous inhibitor for alcohol dehydrogenase is increased after germination. The increase of this inhibitor is concomitant with the decline of alcohol dehydrogenase activity, indicating that the activity of alcohol dehydrogenase during seed germination is controlled by the level of the inhibitor.     

The mechanism of conversion of rat liver xanthine dehydrogenase from an NAD+-dependent form (type D) to an O2-dependent form (type O).

Rat liver xanthine dehydrogenase, type D, has been isolated directly from crude extracts as an antibody complex and its properties compared with those of two oxidase forms of the enzyme, heat-treated type O and trypsin-treated type O, also isolated as antibody complexes. The type D antibody complex displays electron acceptor specificities and electron paramagnetic resonance properties characteristic of an NAD⁺-dependent dehydrogenase whereas the trypsin-treated type O complex behaves as an O₂-utilizing oxidase. The heat-treated type O complex displays intermediate behavior. After electrophoresis in dodecyl sulfate-urea-acrylamide gels, type D and heated type O enzymes show single polypeptide bands, each of approximately 150,000 molecular weight. The trypsinized type O also shows one major band but with an approximate molecular weight of 130,000. Purified type D enzyme, when proteolytically treated, is converted to an oxidase with increased mobility on polyacrylamide gels. The 150,000 molecular weight subunit is cleaved into smaller subunits during proteolysis. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) converts the type D enzyme, whether isolated as the purified enzyme or as the immune precipitate, to type O enzyme in a time-dependent manner. Titration of type D and the two type O antibody complexes with 5,5′-dithiobis-(2-nitrobenzoic acid) reveals that type D and heated type O each has approximately 28 reactive sulfhydryls, whereas the trypsinized type O has only 8 such groups. Many of the free sulfhydryls are vicinal and form disulfide bonds during the conversion to an oxidase by this reagent. Unproteolyzed preparations of type O rat liver enzyme and milk xanthine oxidase are converted to type D enzymes by treatment with dithiothreitol. The converted enzymes display electron acceptor specificities and epr properties characteristic of an NAD⁺-dependent dehydrogenase molecule.     

Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.