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1.
A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD+ oxidoreductase, EC 1.1.1.28] fromLactobacillus helveticus CNRZ32 was identified from a genomic library by complementation ofEscherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kbDraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-) LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci orLactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative ofL. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant. 相似文献
2.
En-Ping Chen Per G. Söderberg Alexander D. MacKerell Jr. Bo Lindström Björn M. Tengroth 《Radiation and environmental biophysics》1989,28(3):185-191
Summary The effect of UV radiation on lactate dehydrogenase (LDH) was investigated. LDH from bovine heart was exposed to 3.6–18 kJ/m2 of ultraviolet (UV) radiation in the 300 nm wavelength region. The activity of LDH was observed to decrease as a function of the dosage of UV radiation. The inactivation of LDH was independent of exposure rate at constant dosage of UV radiation. The decrease of LDH activity caused by UV 300 nm radiation was coupled to a decrease of the maximal velocity (V
max) while the Michaelis constant (K
m
) remained unchanged. The absorption spectrum of the LDH changed during exposure to UV radiation suggesting an alteration of tryptophan in the LDH molecule.On leave from Department of Ophthalmology, Ren-Ji Hospital affiliated to Shanghai No. 2 Medical University, Shanghai, People's Republic of China 相似文献
3.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1764-1766
When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form. 相似文献
4.
Saburova E. A. Avseenko N. V. Simonova N. B. Elfimova L. I. Pronina N. A. Semenenko V. E. 《Russian Journal of Plant Physiology》2000,47(6):761-771
The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V
max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle. 相似文献
5.
In cereal root tissue, hypoxia induces the enzyme lactate dehydrogenase (LDH); (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). In barley, both biochemical and genetic data indicate that five isozymes are induced under hypoxia. These isozymes are tetramers and arise from the random association of the products of two Ldh genes. The induction of LDH activity in root tissue has been shown to be correlated to an increase in LDH protein and Ldh mRNA.In order to more fully characterize the hypoxic induction of LDH, we have isolated a maize Ldh genemic clone which has strong homology at both the amino acid and nucleotide level to the barley LDH cDNA clones. The Ldh1 gene consists of two exons separated by a 296 bp intron, has the expected eukaryotic regulatory signals and a sequence that has strong homology to the maize anaerobic regulatory element. 相似文献
6.
S. Brennan J. Holder J. Baldwin 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,110(4):689-695
Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase Vmax of the pyruvate reductase reaction by 130% to 210%, and decrease Km pyruvate 5 to 11 fold and Km NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation. 相似文献
7.
The LDH isozymes were surveyed in bacterized cultures of syngens 1, 3, 12, and 13 of Paramecium caudatum by polyacrylamide gel electrophoresis. All the examined stocks of different syngens except one stock in syngen 3 had a single common LDH isozyme, and intra- and intersyngenic variation was not observed except for the one stock in syngen 3. Breeding data using the exceptional stock indicated that the LDH isozymes of P. caudatum are controlled by two codominant alleles at a single locus whose products aggregate randomly, forming a dimer. 相似文献
8.
It was found that low oxytetracycline (OTC) concentrations inhibited malic dehydrogenase (MDH) and lactic dehydrogenase (LDH)
inStaphylococcus aureus andEscherichia coli (1–5 μg/ml for MDH and 10 μg/ml for LDH). Inhibition of these enzymes occurred almost instantaneously and could be demonstrated
after 3–4 minutes. No MDH activity was found in OTC-resistant variants of these microorganisms, but LDH activity was not lowered.
The inhibitory effect of OTC is specific for bacterial MDH and LDH. The same enzymes of mammalian origin are not inhibitedin vitro even by high OTC concentrations (100 μg/ml). 相似文献
9.
Yoshikazu Nishiguchi Fumiyoshi Abe Mitsumasa Okada 《Marine biotechnology (New York, N.Y.)》2011,13(2):137-141
The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined.
LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure
of LDH-A4 from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation
of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures. 相似文献
10.
The aim is to investigate the mechanism of miR-499a-5p on the damage of cardiomyocyte induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase (LDH), apoptosis rate and the expression of miR-499a-5p and cluster of differentiation 38 (CD38) in hypoxia-reoxygenation model cells were detected by LDH Cytotoxicity Assay Kit, flow cytometry, real-time polymerase chain reaction, and Western blot analysis, respectively. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p or silencing of CD38 in H9c2 cells. The target relationship between miR-499a-5p and CD38 was verified by Targetscan online prediction and dual-luciferase assay. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p and CD38. Apoptosis, the activity of LDH and the expression of CD38 were increased (P < .05) while expression of miR-499a-5p was decreased (P < .05) in hypoxia/reoxygenation model cells. Apoptosis and the activity of LDH in H9c2 cells after overexpression of miR-499a-5p or silence of CD38 were decreased (P < .05). The results of Targetscan online prediction and dual-luciferase assay indicated that CD38 was a potential target gene of miR-499a-5p. Overexpression of CD38 could reverse the inhibition of miR-499a-5p on LDH activity and apoptosis in H9c2 cells. miR-499a-5p could relief the injury of cardiomyocytes induced by hypoxia/reoxygenation via targeting CD38. 相似文献
11.
The functions of the genes encoded for lactate dehydrogenase (LDH) in six genera of characins (teleost) were examined by electrophoretic and immunochemical analyses of the LDH isozymes. The characins possess the LDH A and B loci present in all vertebrates. The eyeless Mexican cave fish (Anoptichthys jordani) and other characins possessing normal eyes, e.g., Mexican tetra (Astyanax mexicanus, which is able to hybridize with the cave fish), blue tetra (Mimagoniates microlepis), sailfin tetra (Crenuchus spilurus), head and tail light tetra (Hemigrammus ocellifer), and the piranha (Serrasalmus spilopleura), all lack the function of a third LDH locus (the E locus) present in many teleosts which codes for a distinctive isozyme synthesized in the nervous system, particularly in the neural retina.This research was supported by NSF grant GB 16425 to G.S.W. F.S.M. was a James Scholar at the University of Illinois during the course of this research. 相似文献
12.
Studies on the genetic polymorphism of lactate dehydrogenase B (phenotype B−) in rodent erythrocytes
While the common red cell phenotype of lactate dehydrogenase in mammals includes A and B subunits, in some species, especially of the rodent suborder Myomorpha, LDH B subunits are absent in erythrocytes (phenotype B–). A polymorphism involving LDH B+ and LDH B– phenotypes was observed in Mus musculus, and it was found that LDH B– is recessive in relation to LDH B+ (Shows and Ruddle, 1968). A similar polymorphism is described here in the field mouse, Apodemus sylvaticus. In family studies, the recessive mode of inheritance is confirmed. Induction of reticulocytosis reveals that the LDH B– phenotype is not the consequence of degradation of the B subunits in aging red cells. The nature of the mutation giving rise to this polymorphism is discussed.Supported by the Deutsche Forschungsgemeinschaft. 相似文献
13.
Erythrocyte and tissue lactate dehydrogenase (LDH) electrophoretic patterns of 26 rodent species from ten families were examined. The LDH B gene was observed to range in erythrocyte expression from species without detectable B subunits to those which predominantly expressed B subunits. However, the shift in erythrocyte B gene expression was not observed in the tissue LDH electrophoretic patterns between rodent species. Species which did not express erythrocyte B subunits, or only small quantities of B subunits, were restricted to the suborder Myomorpha. In erythrocytes of other rodent species, and most mammals, LDH B subunits are expressed equally or in excess of A subunits. The results suggest either structural differences in the LDH B gene between Myomorph and non-Myomorph rodents or a regulator gene which controls the expression of the B gene in Myomorph erythrocytes. Existing evidence favors the latter hypothesis.Supported by U.S. Public Health Service Grants 5F2 HD-35,531 (T.B.S.), GM-09966 (F.H.R.), G.R.S. 5 SO1 FR-05400-07, and NSF GB 5440X (E.J.M.). 相似文献
14.
A flow injection system for the on-line detection of the intracellular enzyme lactate dehydrogenase (LDH) during fermentation has been developed. The system is comprised of an on-line cell disintegration part, an immobilised dye based expanded bed column for the affinity capture of LDH and a fluorimetric detection unit. The system with a linearity of 0.1–5.4 U LDH ml–1 was applied for the detection of intracellular accumulation of LDH during Lactococcus lactis subsp.lactis cultivation. 相似文献
15.
Cayenne AP Gabert B Stillman JH 《Comparative biochemistry and physiology. Part D, Genomics & proteomics》2011,6(4):393-398
Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein–protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes. 相似文献
16.
Raj Kumar Koiri Surendra Kumar Trigun Santosh Kumar Dubey Santosh Singh Lallan Mishra 《Biometals》2008,21(2):117-126
Metal complex–protein interaction is an evolving concept for determining cellular targets of metallodrugs. Lacatate dehydrogenase
(LDH) is critically implicated in tumor growth and therefore, considered to be an important target protein for anti-tumor
metal complexes. Due to efficient biocompatibility of copper (Cu2+) and zinc (Zn2+), we synthesized CubpyAc2 · H2O (Cu-bpy) and ZnbpyAc2 · H2O (Zn-bpy; where bpy = 2,2′ bipyridine, Ac = CH3COO−) complexes and evaluated their interaction with and modulation of LDH in mouse tissues. The increasing concentration of both
the complexes showed a significant shift in UV–Vis spectra of LDH. The binding constant data (Kc = 1 × 103 M−1 for Cu-bpy and 7 × 106 M−1 for Zn-bpy) suggested that Zn-bpy-LDH interaction is stronger than that of Cu-bpy-LDH. LDH modulating potential of the complexes
were monitored by perfusing the mice tissues with non-toxic doses of Cu-bpy and Zn-bpy followed by activity measurement and
analysis of LDH isozymes on non-denaturing polyacrylamide gel electrophoresis (PAGE). As compared to the control sets, Cu-bpy
caused a significant decline (P < 0.05–0.001) in the activity of LDH in all the tissues studied. However, Zn-bpy showed inhibition of LDH only in liver (P < 0.01), kidney (P < 0.001) and heart (P < 0.01), but with no effect in spleen, brain and skeletal muscle tissues. PAGE analysis suggested that all the five LDH isozymes
are equally sensitive to both the complexes in the respective tissues. The results suggest that Cu- and Zn-bpy are able to
interact with and inhibit LDH, a tumor growth supportive target protein at tissue level. 相似文献
17.
The tolerance of abyssal pressures likely depends on adaptive modifications of fish proteins. However, structural modifications
of proteins which allow functioning at high pressure remain unclear. We compared the activities of lactate dehydrogenase (LDH),
an important enzyme in glycolytic reaction, in three hagfishes inhabiting different depths under increased pressure. LDH in
Eptatretus okinoseanus, found at a depth of 1,000 m, was highly active at high pressure of 100 MPa maintaining the activity at 70% of that at 0.1 MPa.
In contrast, LDH activity in Paramyxine atami, found at 250–400 m, decreased to 55% at 15 MPa, and that in Eptatretus burgeri, found at 45–60 m, was completely absent at 5 MPa. The result suggests that subunit interaction of the LDH-tetramer is more
stable in E. okinoseanus than that in P. atami and E. burgeri under high-pressure conditions. We found six amino acid substitutions between the three LDH primary structures. Accordingly,
these amino acid residues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions. 相似文献
18.
Małgorzata Garnczarska 《Acta Physiologiae Plantarum》2002,24(3):265-272
Seedlings of lupine (Lupinus luteus L. cv. Juno) were exposed for up to 96 hours to 1 to 2 kPa partial pressure oxygen (hypoxic treatment) and activities of alcohol
dehydrogenase (ADH), lactate dehydrogenase (LDH) and their isoform profiles were determined. Roots of lupine seedlings were
grown in a nitrogen flushed nutrient solution while their shoots were in air. Prolonged hypoxia led to a reduction of root
elongation. This was accompanied by reduced increase in dry weight suggesting that insufficient carbohydrate supply was the
cause of retarded growth of lupine roots. Hypoxically treated roots showed induction of ADH and LDH acivities. The maximum
increase in LDH activity was low (2-fold) in contrast to ADH activity, which increased up to 7-fold. Hypoxic treatment of
roots did not affect the activities of ADH and LDH in hypocotyls and cotyledons. Analysis of ADH and LDH activity gels indicated
in roots 1 and 2 isoforms, respectively. The level of isozymes of both enzymes increased in roots upon exposure to hypoxic
stress. Differences in isoenzymatic spectrum of ADH and LDH between roots, hypocotyls and cotyledons indicate organ specificity
of isozymes of both enzymes. The importance of alcohol and lactate fermentation in roots to cope with hypoxic stress is discussed. 相似文献
19.
In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC50 values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.Abbreviations DMSO
dimethyl sulfoxide
- HPC
hepatocyte primary culture
- IC50
50% inhibitory concentration
- LDH
lactate dehydrogenase 相似文献
20.
Over-production of lactate dehydrogenase from Plasmodium falciparum opens a route to new antimalarials 总被引:2,自引:0,他引:2
Dilek Turgut-Balik Debbie K. Shoemark Kathleen M. Moreton Richard B. Sessions J. John Holbrook 《Biotechnology letters》2001,23(11):917-921
Over-production of lactate dehydrogenase (PfLDH) from Plasmodium falciparum from E. coli TG2 cells transformed with a pKK223-3 plasmid containing the wild type gene isolated by Bzik DJ, Fox BA, and Gonyer K (1993) Mol. Biochem. Parasit.
59, 155–166, gave mostly an inactive protein after isolation. Sequencing the N-terminus of the over-produced protein showed that the major product commenced at an internal methionine. Truncation of the protein occurred due to the inappropriate priming from a Shine–Dalgarno (SD) sequence upstream of Met 35. Silent mutations of this SD sequence to remove the purine-rich region allowed over-production of the full length PfLDH up to 15 mg protein l–1 broth. The purified protein exhibited biochemical properties of an authentic LDH enzyme. However, high activity with 3-acetylpyridine adenine dinucleotide as well as with the natural cofactor, NAD, was also observed. The high-resolution X-ray structure obtained from the recombinant enzyme has provided the opportunity for the development of inhibitors specific to PfLDH. 相似文献