Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons

Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin (Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.     

Purification and characterization of glycolate oxidase from pumpkin cotyledons

Glycolate oxidase was purified and crystallized from cotyledons of germinating pumpkin seedlings. The molecular weight of the enzyme was determined to be 280,000-320,000, consisting of 8 identical subunits with molecular weight of 38,000. There are two absorption peaks at 340 and 450 nm, indicating the glycolate oxidase is a flavin protein. Several kinetic parameters were determined, Km (glycolate) 0.33 mM and Km (O2) 76.2 microM at pH 8.0. Oxalate and oxalacetate were found to be potent competitive inhibitors against glycolate; the Ki values for oxalate and oxalacetate were 4.5 and 7.8 mM, respectively. Fatty acids such as linoleic acid inhibited the enzyme noncompetitively; the Km for linoleic acid was 0.63 mM. The regulation of glycolate oxidase in the glycolate pathway occurring in leaf peroxisomes is discussed.     

Alcohol dehydrogenase isoenzymes in chickpea cotyledons

Alcohol dehydrogenase (ADH) (EC 1.1.1.1) in the cotyledons of chickpea consists of three isoenzymes, ADH-1, ADH-2 and ADH-3, in order of decreasing ele     

Purification and characterization of aconitase isoforms from etiolated pumpkin cotyledons

Although aconitase (EC 4.2.1.3) is involved in the glyoxylate cycle, which is localized in glyoxysomes, we detected very low aconitase activity in glyoxysomal fractions after sucrose gradient centrifugation of extracts prepared from etiolated pumpkin ( Cucurbita sp.) colyledons. Two aconitase isoforms were purified to homogeneity, albeit in low yield, by hydrophobic interaction, hydroxylapatite and anion exchange chromatography. They were designated Aco I and Aco II; both were shown to be monomeric proteins of M₁ 100 000 or 98 000 by gel filtration and SDS-PAGE analysis, respectively; isoelectric points were 5.0 and 4.8, respectively. Kinetic studies revealed similarities between Aco I and Aco II. A third aconitase isoform (Aco III) was revealed but not purified to homogeneity.     

Biosynthesis and intracellular transport of glyoxysomal malate dehydrogenase in germinating pumpkin cotyledons

Glyoxysomal malate dehydrogenase was synthesized as a larger molecular mass precursor in germinating pumpkin cotyledons. In pulse-chase experiments, the radioactive larger molecular mass precursor (38 kDa) disappeared and was converted to the mature form (33 kDa) of the enzyme. When the radiolabeled cotyledon was fractionated into cytosolic and organellar fractions, the larger molecular mass precursor was first recovered in the cytosolic fraction and then only after a 20 min chase the mature form was found in the organellar fraction. This indicates that the higher molecular mass precursor is synthesized in the cytosol and the processing of the transient precursor is coupled to the transport into glyoxysomes.     

Glutamate dehydrogenase isoenzymes in Ricinus communis seedlings

Castor bean seedling glutamate dehydrogenase isoenzymes are not artifacts. The isoenzymes have different salting out properties and they utilize NAPD to differing extents, but they have the same isoelectric point of pH 6·2. Tissue specific patterns occur but the patterns are the same between genotypes. The GDH isoenzymes are probably of functional significance in castor bean seedlings.     

Simian liver alcohol dehydrogenase: isolation and characterization of isoenzymes from Macaca mulatta

Like human liver alcohol dehydrogenase, that of Macaca mulatta can be purified and separated into anodic and cathodic pyrazole-insensitive and cathodic pyrazole-sensitive enzyme forms. Their inhibition by 4-methylpyrazole and their substrate specificities are analogous to those observed for the corresponding isoenzymes of human liver. However, on the basis of data available so far, the physiochemical and compositional characteristics, i.e., molecular weight, zinc content, and dimeric structure, of all simian alcohol dehydrogenase forms are virtually identical with those of other mammalian alcohol dehydrogenases studied up to now. Zinc is essential for their enzymatic function, as demonstrated by inhibition with chelating agents.     

NADP+-isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons

NADP⁺-isocitrate dehydrogenase (IDH, EC 1.1.1.42) was studied during the post-germinative growth of pumpkin ( Cucurbita pepo L. cv. Alberello di Sarzana) seedlings. In cotyledons. IDH activity increased in the dark and declined after illumination. Native PAGE showed that at least two isozymes of low electrophoretic mobility are present in cotyledons and absent in other pumpkin tissues. Anion exchange chromatography performed on extracts both from 4-day-old etiolated cotyledons and from illuminated cotyledons confirmed the trend of the IDH isoforms. In senescing cotyledons an additional IDH isoform with higher electrophoretic mobility appears. Overall the data indicate the presence of specific NADP⁺-IDH isoforms in etiolated cotyledons and senescing cotyledons, when glyoxylate cycle enzymes are active. A possible role for these IDH isoforms is proposed.     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications

The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90°C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH₂-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2–13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47 kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75°C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80°C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (V _max of 172U/mg protein), but also utilizes NADPH (V _max of 12U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota. Received: 15 July 1996 / Accepted: 12 October 1996     

Turnover of phytochrome in pumpkin cotyledons

By using density labeling, it was found that the protein moiety of phytochrome is synthesized de novo in the red-absorbing form in cotyledons of dark-grown pumpkin (Cucurbita pepo L.) seedlings, as well as those irradiated with red light and returned to the dark. The rate of synthesis appears to be unaffected by the light treatment. Turnover of the red-absorbing form was also detected in dark grown seedlings using density labeling, while turnover of the far red-absorbing form is already implied from the well known “destruction” observed in irradiated seedlings. In both cases, true degradation of the protein is involved, but the rate constant of degradation of the far red-absorbing form may be up to two orders of magnitude greater than that of the red-absorbing form. The data indicate that, in pumpkin cotyledons, phytochrome levels are regulated against a background of continuous synthesis through divergent rate constants of degradation of the red and far red-absorbing forms and the relative proportions of the two forms present.     

Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulselabeled with [³⁵S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, γ and δ. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg²⁺, and Cu²⁺, but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.     

Glutamate dehydrogenase from Mycoplasma laidlawii

Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H)]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 +/- 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The K(m) for reduced nicotinamide adenine dinucleotide was 1.8 x 10(-4)m. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported.     

Cell-free translation of polyribosomes from detached pumpkin cotyledons: Effects of starvation and cytokinin

The effects of 6-benzylaminopurine (BAP, 5.10^?5M) treatment of pumpkin cotyledons and their starvation after excision upon polysome/monosome ratio and translational capacity of polysomes in cell-free system were studied. It has been found that starvation causes a progressive polysome degradation. Polysome translation in a wheat germ cell-free proteinsynthesizing system reveals that the translation capacity of polysome preparations decreases with the time after cotyledon excision much more sharply than polysome/monosome ratio. This indicates the starvation damage in elongation steps of protein synthesis. The decrease of postribosomal supernatants activity in the system of poly(U)-directed polyphenylalanine synthesis confirms this conclusion. BAP treatment brings about a very rapid monosome mobilization into polysomes and activation of cell-free translation of ribosome preparations which is however closely parallel to the polysome percentage in them. That means that during this initial period of BAP action only protein synthesis initiation is under BAP control. The experiments with aurintricarboxylic acid (ATA) support this idea.     

Cytokinin-modulated gene expression in excised pumpkin cotyledons

Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A)⁺ mRNA isolated from cotyledons incubated with 10⁻⁴ molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10⁻⁹ to 10⁻⁴ molar) of benzyladenine. Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxypyruvate reductase activity; thus, the enzyme synthesis does not appear to be controlled by organelle genes. Hydroxypyruvate reductase synthesis is inhibited by cycloheximide, cordycepin, and to a certain degree by actinomycin D. These data support the view of a close association between cytokinin action and gene expression.     

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.     

Malate dehydrogenase isoenzymes from cotton leaves: molecular weights

Malate dehydrogenase isolated from leaves of the cotton plant (Gossypium hirsutum L.) appears in the form of several isoenzymes. Four of the isoenzymes found in cotton leaf extracts appear to be charge isomers with a molecular weight of approximately 60,000. A fifth malate dehydrogenase isoenzyme found in leaf extracts has a molecular weight of approximately 500,000. Under appropriate conditions it is possible to form this high molecular weight isoenzyme from at least one of the smaller isoenzymes. In addition, malate dehydrogenase isoenzymes of approximately 700,000 and 130,000 molecular weight have been observed under some conditions, although these isoenzymes do not appear in the crude cotton leaf preparations. The relationship of this heterogeneity with respect to size and to the discrepancies in the number and size of malate dehydrogenase isoenzymes reported from plant tissues may be significant.