Regulation of biosynthesis of secondary metabolites

Two partly purified malate dehydrogenase (EC 1.1.1.37) isoenzymes were isolated fromStreptomyces aureofaciens. This is the first example of a non-homogeneous enzyme in actinomycetes and one of the very few cases in bacteria in general. The characteristics of the enzymatic reaction were studied for each enzyme in relation to the concentration of both substrates and cofactors and the apparent Michaelis constant was calculated. It was found that the reaction was affected by Mg²⁺ ions and that SH-groups could be specifically inhibited. The optimal pH and the influence of temperature changes were also determined. In all the parameters, one of the isoenzymes resembled mitochondrial MDH, while the other resembel the supernatant MDH described in the literature in the tissues of higher organisms. The functional relationship of the two MDH isoenzymes inStreptomyces aureofaciens is discussed.     

Multiple forms of NAD-dependent malate dehydrogenase in 3 species of trematodes of the genus Notocotylus (Trematoda: Notocotylidae)

NAD dependent malate dehydrogenases of three trematode species, Notocotylus attenuatus, N. ephemera and N. imbricatus, have been investigated by electrophoresis. Seven different zones with 15 isoenzymes in N. attenuatus, 16 isoenzymes in N. ephemera and 11 isoenzymes in N. imbricatus have been found in MDH spectra. Isoenzymes of MDH are controlled by seven polymorphic loci. The activity of isoenzymes of three slowly migrating zones is 10 and more times higher than that of fast zones (4-7). The genotypes of adults in one strain are genetically identical, independent ot the development in different definitive hosts. The spectra of MDH of the investigated Notocotylus species are different in slowly migrating isoenzymes (1-3 zones).     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.     

Differential Regulation of Malate Dehydrogenase Isoenzymes by Hydrocortisone in the Liver and Brain of Aging Rats

The activities and induction patterns of the isoenzymes of malate dehydrogenase (MDH) of the liver and brain of male rats of various ages were studied. The activities of both the isoenzymes of MDH of the liver and brain show a gradual increase with increasing age of the rats. Adrenalectomy decreases and hydrocortisone treatment increases the activity of cytoplasmic MDH of the liver and brain of rats of all the ages except that of the brain isoenzyme of old rats. This hormone-mediated induction of the isoenzyme is actinomycin D-sensitive. Furthermore, adrenalectomy decreases and hydrocortisone treatment increases the activity of mitochondrial MDH of the liver of young and adult rats but not in old rats. However, these treatments do not show any significant effect on the activity of mitochondrial MDH of the brain of rats of all the ages.     

Soluble and chloroplast malate dehydrogenase isoenzymes of Triticum aestivum

Three isoenzymes of malate dehydrogenase have been isolated from 9-day-old wheat shoots. The microbody (peroxisome) and chloroplast MDH are similar in their electrophoretic behaviour. The mitochondrial MDH, soluble MDH and chloroplast MDH differ in K_m values for malate and NAD. The activity of MDH isoenzymes with NAD⁺-analogues as substrate was in the order 3-AP-NAD⁺ > 3-AP-deam NAD⁺ > NAD⁺ > TN-NAD⁺ and deam NAD⁺. The thermal stabilities of the isoenzymes were significantly different: C-MDH > m-MDH > S-MDH.     

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.     

Cd2+胁迫对小麦幼苗生长及呼吸作用的影响

以小麦品种郑州9023为材料,研究了不同浓度Cd2 胁迫对小麦幼苗生长及呼吸作用的影响.结果显示:(1)随Cd2 胁迫浓度的升高,小麦幼苗根和芽的呼吸速率及琥珀酸脱氢酶(SDH)活性均呈先上升后下降的趋势.(2)Cd2 胁迫对小麦幼苗根中细胞色素氧化酶(COD)、苹果酸脱氢酶(MDH)、异柠檬酸脱氢酶(IDH)同工酶表达的影响较小,都呈低浓度诱导、高浓度抑制的效应,且Cd2 处理诱导了根中新的MDH、IDH同工酶带的表达;而不同浓度Cd2 对小麦幼苗芽中COD、MDH、IDH同工酶的表达影响较小.(3)随Cd2 胁迫浓度的增加,芽长、根长、芽干重、根干重均呈持续下降的趋势,且对根的抑制作用明显大于对芽.研究表明,Cd2 胁迫可以改变小麦幼苗根和芽中SDH、COD、MDHI、DH等呼吸作用关键酶的活性或同工酶表达,从而影响其呼吸作用,最终抑制了幼苗的生长.     

Changes in lactate and malate dehydrogenase isoenzymes in salmonella under the effect of potassium dichloroisocyanurate

The method of enzyme-electrophoresis in agar gel according to Wieme (1959) was used for the study of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes of 24-hour and 48-hour Salmonella cultures exposed to a 0.02% solution of potassium dichloroisocyanurate (PDIC). Severe repression of LDH and MDH isoenzymes was observed immediately after the exposure of the culture to the disinfectant solution. A significant decrease in the content of the isoenzyme LDH1 and of the cytoplasmic fraction (C1) of MDH simultaneously with the appearance of the fractions LDH4, LDH1a and LDH1b were established in the strains cultured on MPA in the course of 24 hours following the exposure. A tendency to a decrease in the LDH1 content was preserved in the experimental cultures after 48 hours, but the spectrum of MDH isoenzymes showed almost no differences in comparison with that of MDH isoenzymes in 48-hour cultures of the control strains.     

Induction of a Peroxisomal Malate Dehydrogenase Isoform in Liver of Starved Rats

The influence of starvation on malate dehydrogenase (MDH) in rat liver was investigated. Native electrophoresis revealed two MDH isoforms in non-starved rats and three isoenzymes in starved rats. After sucrose density gradient centrifugation of cell organelles from liver, MDH activity was detected in the mitochondrial and cytosolic fractions from non-starved rats. However, additional activity was found in the peroxisomal fraction from starved rats. The latter was identified as the electrophoretically new isoform in starved animals. The three isoforms of malate dehydrogenase from hepatocytes were separated and partially purified by chromatography on DEAE-Toyopearl. Several kinetic and regulatory properties of the three isoforms were rather similar. It is suggested that the newly expressed isoform of MDH operates in the glyoxylate cycle of liver peroxisomes of food-starved animals.     

Glycolytic enzymes in polyamine-treated bovine retina

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.     

Sucrose feeding prevents changes in myosin isoenzymes and sarcoplasmic reticulum Ca2+-pump ATPase in pressure-loaded rat heart

Pressure-overload due to banding of the abdominal aorta in rats for 10 weeks resulted in cardiac hypertrophy, redistribution of myosin isoenzymes and reduction in the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase activity. Administration of sucrose in the drinking water (0.8%, w/v) to rats prevented changes in myosin isoenzymes and SR Ca2+-stimulated ATPase in hypertrophied hearts. This beneficial effect of sucrose feeding with respect to remodeling of the subcellular organelles in the myocardium was not associated with any significant changes in plasma glucose or thyroid hormone levels. It is suggested that the prevention of subcellular changes in the hypertrophied hearts due to sucrose feeding may be due to a shift in fuel utilization by the myocardium.     

Malate dehydrogenase of a mosquito,Culex p. quinquefasciatus: Developmental changes,polymorphism, and physicochemical characterization

Malate dehydrogenase (MDH) of larval, pupal, and adult stages of Culex p. quinquefasciatus has been characterized by electrophoresis, isoelectric focusing, and other physicochemical means. It exists as a multiple molecular form possessing a large number of isoenzymes, from a minimum of three in early instar larvae to as many as 14 in adults. The isoenzyme pattern changes during development with respect to both relative activity and the appearance of some new forms and disappearance of others. Each developmental stage possesses a characteristic electrophoretic and gel isoelectric focusing pattern. MDH isoenzymes differ in their response to heat and thiol reagents. Similar electrophoretic variants from larvae, pupae, and adults show great differences in their response to heat treatment at 50 C and 56 C, indicating some differentiation of isoenzymes in each stage of development. Homogenization of whole mosquitos in mercaptoethanol solution results in a sharp increase in the activity of the principal bands and a decrease or disappearance of minor ones. The possibility of some minor bands being "conformers" arising due to nongenetic factors is discussed.     

鳙团移核鱼LDH,MDH同工酶的研究

对二龄鳙鱼细胞核和团头鲂细胞质配合的核质杂种鱼－－鳙团移核鱼及其亲本,供核体鳙鱼和受核体团头鲂肌组织ＬＤＨ、ＭＤＨ同工酶进行了研究试验。鳙团移核鱼和供核体鳙鱼肌组织ＬＤＨ同工酶均具有ＬｄｈＡ２Ｂ２一条谱带;受核体团头鲂的则具有ＬｄｈＡ２、ＬｄｈＡ２Ｂ１、ＬｄｈＡ２Ｂ２、ＬｄｈＡ１Ｂ３、ＬｄｈＢ４等五条谱带。移核鱼和供核体鳙鱼肌组织的ＭＤＨ同工酶都各具有二条谱带：Ｓ－ｍｄｈＡ２、Ｓ－ｍｄｈＡＢ;受核     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.     

Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase.

The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.     

Purification and properties of cytoplasmic and mitochondrial malate dehydrogenases of Physarum polycephalum

Two isoenzymes of malate dehydrogenase (MDH) were demonstrated in plasmodia of Physarum polycephalum by polyacrylamide-gel electrophoresis. The more "cathodal" form was uniquely associated with mitochondria (M-MDH) and the other form was found in the soluble cytoplasm (S-MDH). The isoenzymes were separated by acetone fractionation of soluble plasmodial homogenates acidified to pH 5.0. The M-MDH was purified 201-fold by cetylpyridinium chloride treatment, fractionation with ammonium sulfate, gradient elution from sulfoethyl cellulose at pH 6.0, and Sephadex G-100 chromatography. The S-MDH was purified 155-fold by ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gradient elution from sulfoethyl cellulose at pH 5.5, and Sephadex G-100 chromatography. The optimal cis-oxalacetate concentrations were 0.35 mM for M-MDH and 0.25 mM for S-MDH, and the optimal pH for both isoenzymes was 7.6 for oxalacetate reduction. The optimal l-malate concentrations were 5 mM for S-MDH and 6 mM for M-MDH, and both isoenzymes exhibited an optimal pH of 10.0 for L-malate oxidation. The Michaelis constants of S-MDH and M-MDH served to discriminate between the isoenzymes. The S-MDH was more heat-stable than the M-MDH. High concentrations of oxalacetate and malate inhibited S-MDH more than M-MDH. The isoenzymes were further distinguished by their utilization of analogues of nicotinamide adenine dinucleotide. Many properties of the Physarum isoenzymes were similar to those of more complex organisms, especially vertebrates.     

Determination of enzyme mechanisms by molecular dynamics: studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenase

Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH- [-HC5(O-)-C4(=O)-] and PQQH [-HC5(OH)-C4(=O)-] converts to the hydroquinone PQQH2 [-C5(OH)=C4(OH)-]. The main focus is on MD structures of MDH * PQQ * methanol, MDH * PQQH-, MDH * PQQH, sGDH * PQQ * glucose, sGDH * PQQH- (glucolactone, and sGDH * PQQH. The reaction PQQ-->PQQH- occurs with Glu 171-CO2- and His 144-Im as the base species in MDH and sGDH, respectively. The general-base-catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen-bonding to the C5=O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297-COOH would act as a proton donor for the reaction PQQH(-)-->PQQH, if formed by transfer of the proton from Glu 171-COOH to Asp 297-CO2- in MDH. For PQQH-->PQQH2, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen-bonded to Glu 171-CO2- in MDH and by Wat89 in sGDH).     

Molecular,biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.     

Cytosol malate dehydrogenase in Calicophoron ijimai trematodes and the effect of antiparasitic preparations on its activity

The activity and properties of malate dehydrogenase (MDH; EC 1.1.1.37) and of "malic" enzyme (EC 1.1.1.40) in cytosole of the trematode C. ijimai were determined. The activity of MDH directed to oxaloacetate formation was shown to be 14 times and maximum velocity 13 times lower than that of the reverse reaction. The apparent KM was one order higher in the direct reaction. This confirms the possibility of glycolytic pathway in C. ijimai via CO2 fixation into phosphoenolpyruvate to form oxaloacatate which is readily eliminated by active MDH. The presence of "malic" enzyme in C. ijimai testifies to the occurrence of different pathways of succinate formation in this species.