The osmotic lysis of spiroplasma cells and its use in enzyme studies

1994 ◽  
Vol 40 (9) ◽  
pp. 791-794 ◽  
Author(s):  
Jianchi Chen ◽  
C. J. Chang
Keyword(s):  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Citrate Synthase ◽  
Nadh Oxidase ◽  
Tricarboxylic Acid Cycle ◽  
Spiroplasma Citri ◽  
Cell Lysates ◽  
Wash Buffer ◽  
Osmotic Lysis ◽  
Spiroplasma Melliferum

A simple and efficient osmotic lysis method was developed for enzyme studies in spiroplasmas. Log phase cells in R2 medium were harvested by centrifugation (19 600 × g for 30 min). Wash buffer supplemented with 0.23 M sucrose maintained the helicity of spiroplasma cells during washing. Osmotic lysis of spiroplasmas was achieved in H buffer that contained no sucrose. Sucrose at concentrations as low as 0.004 M dramatically increased the resistance of the spiroplasmas to osmotic lysis. NADH oxidase, lactate dehydrogenase, and malate dehydrogenase were detected in cell lysates of Spiroplasma floricola (23-6), Spiroplasma citri (R8A2), Spiroplasma apis (SR 3), and Spiroplasma melliferum (AS 576). Citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl coenzyme A synthetase, succinate dehydrogenase, and fumarase were not detected in cell lysates of S. floricola (23-6). NADH oxidase and malate dehydrogenase were found in the cytosol whereas lactate dehydrogenase was loosely associated with the cytomembrane.Key words: spiroplasmas, osmotic lysis, tricarboxylic acid cycle, enzymes.

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

pH-mediated control of anti-aggregation activities of cyanobacterial and E. coli chaperonin GroELs

2020 ◽  
Author(s):  
Tahmina Akter ◽  
Hitoshi Nakamoto
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Citrate Synthase ◽  
Ph Dependence ◽  
Chaperone Activity ◽  
Ph Change ◽  
Aggregation Assay ◽  
E Coli ◽  
Aggregation Activity

Abstract In contrast to Escherichia coli, cyanobacteria have multiple GroELs, the bacterial homologues of chaperonin/Hsp60. We have shown that cyanobacterial GroELs are mutually distinct and different from E. coli GroEL with which the paradigm for chaperonin structure/function has been established. However, little is known about regulation of cyanobacterial GroELs. This study investigated effect of pH (varied from 7.0 to 8.5) on chaperone activity of GroEL1 and GroEL2 from the cyanobacterium Synechococcus elongatus PCC7942 and E. coli GroEL. GroEL1 and GroEL2 showed pH dependency in suppression of aggregation of heat-denatured malate dehydrogenase, lactate dehydrogenase and citrate synthase. They exhibited higher anti-aggregation activity at more alkaline pHs. Escherichia coli GroEL showed a similar pH-dependence in suppressing aggregation of heat-denatured lactate dehydrogenase. No pH dependence was observed in all the GroELs when urea-denatured lactate dehydrogenase was used for anti-aggregation assay, suggesting that the pH-dependence is related to some denatured structures. There was no significant influence of pH on the chaperone activity of all the GroELs to promote refolding of heat-denatured malate dehydrogenase. It is known that pH in cyanobacterial cytoplasm increases by one pH unit following a shift from darkness to light, suggesting that the pH-change modulates chaperone activity of cyanobacterial GroEL1 and GroEL2.

scholarly journals Identification of a multienzyme complex of the tricarboxylic acid cycle enzymes containing citrate synthase isoenzymes from Pseudomonas aeruginosa

1996 ◽  
Vol 313 (3) ◽  
pp. 769-774 ◽  
Author(s):  
Colin G. MITCHELL
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Growth Cycle ◽  
Tricarboxylic Acid ◽  
Multienzyme Complex ◽  
Acid Cycle ◽  
Consecutive Reactions ◽  
Osmotic Lysis ◽  
The Individual

A multienzyme complex of tricarboxylic acid cycle enzymes, catalysing the consecutive reactions from fumarate to 2-oxoglutarate, has been identified in extracts of Pseudomonas aeruginosa prepared by gentle osmotic lysis of the cells. The individual enzyme activities of fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase can be used to reconstitute the complex. The citrate synthase isoenzymes, CSI and CSII, from this organism can be used either together or as the individual activities to reconstitute the complex. No complex can be reformed in the absence of CSI or CSII. Which CS isoenzyme predominates in the complex depends on the phase of growth at which the cells were harvested and the extract prepared. More CSI was found in the complex during exponential growth, whereas CSII predominated during the stationary phase. The results support the idea of a ‘metabolon’ in this organism, with the composition of the CS component varying during the growth cycle.

STUDIES ON SOME OF THE ENZYMES OF CARBOHYDRATE METABOLISM AND THE CHEMICAL COMPOSITION OF HEN TIBIA

1967 ◽  
Vol 45 (3) ◽  
pp. 387-394
Author(s):  
H. R. Chokshi ◽  
C. V. Ramakrishnan
Keyword(s):  
Lactate Dehydrogenase ◽  
Chemical Composition ◽  
Glutamine Synthetase ◽  
Isocitrate Dehydrogenase ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Fumarate Hydratase ◽  
The Other ◽  
Aconitate Hydratase ◽  
Calcium Phosphorus

Periosteum, cartilage, epiphyseal head, cortical shaft, and marrow of the tibia of White Leghorn hen were studied for chemical composition with regard to calcium, phosphorus, nitrogen, hexosanaine, and moisture, and the cartilage, epiphyseal head, and cortical shaft were also analyzed for citric acid content. In cortical shaft, cartilage, and epiphyseal head the calcium, phosphorus, and hexosamine contents were higher and the nitrogen was lower than in periosteum and marrow. The former parts also showed a greater degree of calcification as judged by Ca:P and Ca:N ratios. Studies were made on the distribution of key enzymes of glycolysis, of the tricarboxylic acid cycle, the hexose monophosphate shunt, and of certain other enzymes in those parts of the tibia that were studied. These enzymes were α-glucan phosphorylase, lactate dehydrogenase, citrate synthase, aconitate hydratase, isocitrate dehydrogenase (NADP), glucose-6-phosphate dehydrogenase, fumarate hydratase, glutamine synthetase, hexosamine synthetase, and L-aspartate: 2-oxogiutarate aminotransferase.The activity of lactate dehydrogenase was found to be much higher than that of the other enzymes in all the regions studied, which strengthens the hypothesis of a greater degree of glycolysis in bone.The activities of aconitate hydratase, isocitrate dehydrogenase (NADP), fumarate hydratase, and lactate dehydrogenase were lower in cortical shaft than in other regions of the bone. Glutamine synthetase was found in marrow but not in the other parts studied. In general, the marrow was found to be enzymically more active than the other regions.

Tricarboxylic Acid Cycle Enzymes of the Ectomycorrhizal Basidiomycete, Suillus bovinus

2001 ◽  
Vol 56 (5-6) ◽  
pp. 334-342 ◽  
Author(s):  
Norbert Grotjohann ◽  
Yi Huangb ◽  
Wolfgang Kowallik
Keyword(s):  
Succinate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Technical Problems ◽  
Acid Cycle ◽  
Suillus Bovinus ◽  
Cell Extracts

In crude cell extracts of the ectomycorrhizal fungus, Suillus bovinus, activities of citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase have been proved and analyzed. Citrate synthase exhibited high affinities for both its substrates: oxaloacetate (Km = 0.018 mᴍ) and acetyl-CoA (Km = 0.014 mᴍ) . Aconitase showed better affinity for isocitrate (Km = 0.62 mᴍ) than for citrate (Km = 3.20 mᴍ) . Analysis of isocitrate dehydrogenase revealed only small maximum activity (60 nmol x mg protein-1 x min -1), the enzyme being exclusively NADP+-dependent. Using the artificial electron acceptor dichlorophenol indophenol, activity and substrate affinity of succinate dehydrogenase were rather poor. Fumarase proved Fe2+-independent. Its affinity for malate was found higher ( Km = 1.19 mᴍ) than that for fumarate ( Km = 2.09 mᴍ) . High total activity of malate dehydrogenase could be separated by native PAGE into a slowly running species of (mainly) cytosolic (about 80%) and a faster running species of (mainly) mitochondrial origin. Affinities for oxaloacetate of the two enzyme species were found identical within limits of significance (Km = 0.24 mᴍ and 0.22 mᴍ) . The assumed cytosolic enzyme exhibited affinity for malate (Km = 5.77 mᴍ) more than one order of magnitude lower than that for oxaloacetate. FPLC on superose 12 revealed only one activity band at a molecular mass of 100 ± 15 kDa. Activities of 2-oxoglutarate dehydrogenase and of succinyl-CoA synthetase could not be found. Technical problems in their detection, but also existence of an incomplete tricarboxylic acid cycle are considered. Metabolite affinities, maximum activities and pʜ-dependences of fumarase and of malate dehydrogenase allow the assumption of a reductive instead of oxidative function of these enzymes in vivo.

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

AbstractMitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

Chronic stimulation of mammalian muscle: enzyme changes in individual fibers

1986 ◽  
Vol 251 (4) ◽  
pp. C633-C642 ◽  
Author(s):  
M. M. Chi ◽  
C. S. Hintz ◽  
J. Henriksson ◽  
S. Salmons ◽  
R. P. Hellendahl ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Adenylate Kinase ◽  
Citrate Synthase ◽  
Single Fiber ◽  
Staining Reaction ◽  
Type I ◽  
Fructose Bisphosphatase ◽  
Chronic Stimulation ◽  
Coa Transferase

Single fibers of rabbit fast-twitch tibialis anterior (TA) muscles were analyzed after continuous low-frequency stimulation for up to 8 wk. After 2-5 wk, every fiber showed higher levels of citrate synthase, hexokinase, and 3-oxoacid CoA-transferase than any control fiber; in some cases these levels were 2-10 times higher (well above any found even in the control soleus, a slow-twitch muscle). Average levels of malate dehydrogenase and alanine transaminase also rose dramatically, but peak single fiber levels were not much above the highest in controls. These differential effects confirm at the single fiber level that chronic stimulation can alter mitochondrial composition. Lactate dehydrogenase, fructose-bisphosphatase, and adenylate kinase declined to levels far below those of any control TA fiber, and, in the case of fructose-bisphosphatase, to within the activity range of control soleus fibers. According to their staining reaction for myofibrillar ATPase, TA fibers were initially 23% type IIA, and 74% type IIB, but by 5 wk these had been converted to a mixture of type I, IIA, and IIC fibers. At 5 wk, levels of lactate dehydrogenase, adenylate kinase, and malate dehydrogenase were characteristic of their (new) ATPase type, but 3-oxoacid CoA transferase had increased to levels 6-15 times higher than in control fibers of the same type.

scholarly journals Four Weeks of Aerobic Training Affects Cardiac Tissue Matrix Metalloproteinase, Lactate Dehydrogenase and Malate Dehydrogenase Enzymes Activities, and Hepatorenal Biomarkers in Experimental Hyperhomocysteinemia in Rats

2021 ◽  
Vol 22 (13) ◽  
pp. 6792
Author(s):  
Dusan Todorovic ◽  
Marija Stojanovic ◽  
Ana Medic ◽  
Kristina Gopcevic ◽  
Slavica Mutavdzin ◽  
...  
Keyword(s):  
Physical Activity ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Subcutaneous Injection ◽  
Cardiac Tissue ◽  
Albino Rats ◽  
Wistar Albino Rats ◽  
And Control ◽  
Tissue Matrix ◽  
Increased Activity

The aim of this study was to investigate the effect of the application of homocysteine as well as its effect under the condition of aerobic physical activity on the activities of matrix metalloproteinases (MMP), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in cardiac tissue and on hepato-renal biochemical parameters in sera of rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C: 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.); H: homocysteine 0.45 µmol/g b.w./day s.c.; CPA saline (0.9% NaCl 0.2 mL/day s.c.) and a program of physical activity on a treadmill; and HPA homocysteine (0.45 µmol/g b.w./day s.c.) and a program of physical activity on a treadmill. Subcutaneous injection of substances was applied 2 times a day at intervals of 8 h during the first two weeks of experimental protocol. Hcy level in serum was significantly higher in the HPA group compared to the CPA group (p < 0.05). Levels of glucose, proteins, albumin, and hepatorenal biomarkers were higher in active groups compared with the sedentary group. It was demonstrated that the increased activities of LDH (mainly caused by higher activity of isoform LDH2) and mMDH were found under the condition of homocysteine-treated rats plus aerobic physical activity. Independent application of homocysteine did not lead to these changes. Physical activity leads to activation of MMP-2 isoform and to increased activity of MMP-9 isoform in both homocysteine-treated and control rats.

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