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1.
Fluorescence titration has been used to determine the binding constant and number of binding sites for the textile triazine dye Procion Yellow HE-3G to lactate dehydrogenase from rabbit muscle (E.C. 1.1.1.27). Triazine dye was either free in solution or attached to one of the polymer carriers, polyethylene glycol or dextran. Titrations were performed in solutions of buffer, dextran, and polyethylene glycol. Aqueous two-phase systems composed of polyethylene glycol and dextran were prepared and the binding constant and number of binding sites for ligand polyethylene glycol-Procion Yellow to lactate dehydrogenase were determined in both upper and lower phases of these systems. Affinity partition of lactate dehydrogenase in a PEG-dextran system was also performed using PEG-Procion Yellow as ligand, and partition coefficients of lactate dehydrogenase showed good agreement with theoretical partition coefficients calculated from the binding constant and number of binding sites obtained from fluorescence titration. The advantage of using fluorescence titration to determine affinity of a polymer ligand for a protein is that measurement of binding strength can be made in the actual environment encountered by protein-ligand complex during the purification process. 相似文献
2.
A simple methoxylated derivative of the triazine dye, Procion blue H-B, selectively precipitates rabbit muscle lactate dehydrogenase from solution. Optimum protein precipitation occurred at an enzyme subunit:dye ratio of approximately 2:1 and was fully reversible upon addition of competitive ligands such as NADH. With a crude extract of rabbit muscle, affinity precipitation with the dye followed by dissolution with NADH yielded homogeneous lactate dehydrogenase in 97% overall yield. 相似文献
3.
《Enzyme and microbial technology》1987,9(4):233-237
Extraction between two aqueous phases has been used for rapid partial purification of enzymes present in porcine muscle using polymer-bound triazine dyes. These enzyme ligands, bound to poly(ethylene glycol), are restricted to the upper phase of a water-dextran-poly(ethylene glycol) two-phase (liquid-liquid) system. Nineteen triazine dyes were tested for their abilities to extract some enzymes (lactate dehydrogenase, malate dehydrogenase, myokinase and pyruvate kinase) present in crude muscle sap into the dye-containing upper phase. Bulk proteins were to large extent recovered in the lower dextran-rich phase. By sequential change of the ligand several of the studied enzymes were extracted into separate upper phases and in 2–8 times purification (relative to protein) within 25 min. The two dehydrogenases were, however, extracted together. The total time for enzyme preparation was reduced to 40 min by direct homogenization of the tissue in the liquid-liquid two-phase system followed by five affinity extraction steps and separation of enzyme from the dye ligands. The yield was in this case considerably reduced. 相似文献
4.
The tetrameric molecule of pig skeletal muscle lactate dehydrogenase binds a cationic fluorescent probe, auramine O, at four equal non-interacting sites with a dissociation constant of (1.25 +/- 0.2) X 10(-4) M. Fluorescence of the dye/enzyme mixture is strongly pH-dependent, with a maximum at pH 6.3-6.8. Auramine O-binding sites are located outside the active center of the enzyme. The microenvironment of the bound dye changes upon interaction of lactate dehydrogenase with NAD+, NADH, ADP and pyruvate. The binding of specific ligands induces an increase in fluorescence of auramine O-enzyme complex. This effect was used to determine the dissociation constants of the complexes of lactate dehydrogenase with specific ligands. Pyruvate was demonstrated to bind to the apoenzyme-auramine O complex with a dissociation constant of 5.2 X 10(-4) M. With the use of auramine O, it became possible to reveal subunit interactions within the tetrameric molecule of lactate dehydrogenase. They are manifested in the changes of the microenvironment of a dye-binding site located on one of the subunits induced by the binding of ligands in the active center of a neighboring subunit. 相似文献
5.
Kuzon WM Aydin MA Green HJ Rosenblatt JD Pynn BR Plyley MJ McKee NH 《Plastic and reconstructive surgery》2004,113(3):932-941
A canine gracilis model was used to study muscle energy metabolism and enzyme activities after free vascularized muscle transfer. Fifteen male mongrel dogs underwent orthotopic, free transfer of the left gracilis with microneurovascular anastomosis. After a minimum of 10 months' recovery, muscle biopsy specimens were obtained from the transfers and the contralateral controls and analyzed for relative fiber type areas and maximum activities of phosphorylase, hexokinase, phosphofructokinase, glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, 3-hydroxyacyl coenzyme A dehydrogenase (HAD), and creatine phosphokinase. Biopsy specimens obtained before and after a 10 minute, 20-Hz contraction were analyzed for glucose, glycogen, glycolytic intermediates, phosphocreatine, total creatine, and adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, and inosine). There was no significant transfer versus control difference in type I relative fiber area (45 +/- 4 percent versus 44 +/- 3 percent). Total creatine was significantly reduced in the transferred muscles relative to control (83.1 +/- 3.0 mmol/kg versus 100.6 +/- 5.1 mmol/kg dry weight). Maximal activities of phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, HAD, and creatine phosphokinase were diminished in transfers relative to controls, although hexokinase activity was significantly higher in the freely transferred gracilis muscles. During the 20-Hz contraction, muscle transfers produced less force initially, although the force/time integral over the 10-minute stimulation was similar in transfers (277 +/- 25 N/g/second) and controls (272 +/- 24 N/g/second). The contraction was associated with significant glvcogen use and lactate accumulation in both transfers and controls, although this was less pronounced for the transfers. Glycolytic flux appeared muted in the transfers relative to controls. Significant, similar high-energy phosphagen reductions and inosine monophosphate accumulation were noted during the contraction in both groups. Contractile activity is associated with the expected pattern of muscle metabolite changes following free vascularized transfer, indicating the components of cellular energy metabolism are not qualitatively altered after microneurovascular muscle transfer. In contrast, quantitative differences suggest that free vascularized muscle transfer can be associated with a muscle enzyme profile consistent with deconditioning and the presence of denervated muscles fibers in the absence of fiber type profile changes. 相似文献
6.
Affinity extraction of dye- and metal ion-binding proteins in polyvinylpyrrolidone-based aqueous two-phase system 总被引:1,自引:0,他引:1
Affinity extraction of dye- and metal ion-binding proteins, respectively, in a polyvinylpyrrolidone (PVP40)-Reppal PES 100 two-phase system was investigated. Due to the ability of PVP to complex azo dyes and inorganic ions, covalent coupling of the ligands was not essential. Cibacron Blue F3GA was used as the ligand for extraction of lactate dehydrogenase (LDH) from porcine muscle, while copper ions were used for extraction of B. stearothermophilus LDH with a fusion tag of six histidine residues (His6-LDH) from recombinant Escherichia coli homogenate. The binding strength of the enzymes to their respective ligands was only slightly reduced in the presence of PVP. The partition coefficient of Cibacron Blue and Cu2+ ions in the two-phase systems composed of different concentrations of PVP and Reppal was in the range of 20-30, with maximal partitioning being observed in the 17% (w/w) PVP40-10% Reppal PES100 system. Only a minor leakage of the ligands to the bottom phase was observed with time. The partitioning of porcine LDH to the PVP phase was increased 100-fold, and a maximal recovery of 89% was obtained in the two-phase system loaded with 0.2% (w/w) Cibacron Blue. The enzyme was quantitatively recovered with further purification from the PVP-dye phase using a secondary extraction step with 170 mM phosphate or alternatively with 100 mM phosphate containing NADH or NaCl. A more than 10-fold increase in the partition coefficient of His6-LDH was achieved in the two-phase system loaded with 0.4% (w/w) copper sulfate compared to the system lacking the metal ions. The enzyme was also back-extracted into phosphate phase in the presence of imidazole. 相似文献
7.
Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining 总被引:2,自引:0,他引:2
C S Hintz E F Coyle K K Kaiser M M Chi O H Lowry 《The journal of histochemistry and cytochemistry》1984,32(6):655-660
Fibers in cross sections of human and rat muscle were typed by using histochemical ATPase stains, and the results were compared with those of quantitative enzyme assays of fragments of the same fibers dissected from serial freeze-dried sections. Two enzymes previously used to assess the metabolic type were measured in each case: lactate dehydrogenase and either adenylokinase (human fibers) or malate dehydrogenase (rat fibers). With human fibers there was essentially complete agreement between ATPase staining and the metabolic enzyme assays in distinguishing types I and II fibers. The agreement was less consistent with regard to type IIA and IIB fibers. A number of ATPase type IIC fibers were identified in one human muscle, and were found to fall between ATPase types I and IIA on the basis of metabolic enzyme assay results. Rat-fiber ATPase types I, IIA, and IIB from the plantaris muscle were rather well segregated on a two-dimensional lactate dehydrogenase-malate dehydrogenase grid. In the rat soleus muscle, ATPase types I and IIA fibers were shifted to lower lactate dehydrogenase levels, with IIC fibers interposed between them. 相似文献
8.
M V Sukhodolets V I Muronetz N K Nagradova 《Biochemical and biophysical research communications》1989,161(1):187-196
An interaction of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled with FITC was studied by following the changes in fluorescence intensity of the bound dye. The association between the two enzymes was found to be a rather slow process characterized by a second order rate constant of 1.1 +/- 0.2.10(3) M-1 s-1, the KD of the complex between apoenzymes being 3.2.10(-7) M. The stability of the complex increased upon increase of temperature and ionic strength of the medium, suggesting a hydrophobic character of association. The ligands which bind at the active centers of the two enzymes (NAD+, ATP, 3-phosphoglycerate) weakened the bienzyme association. Unlabeled 3-phosphoglycerate kinase was unable to displace the FITC-labeled enzyme from the complex. Taken together, the results indicate that interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by FITC is assisted by the dye, which may bind at nucleotide-binding sites of GPDH. No interaction was observed between the FITC-labeled 3-phosphoglycerate kinase and lactate dehydrogenase, which suggests that protein-protein interaction at specific "recognition" sites may be a prerequisite for the complex formation. 相似文献
9.
S. B. Chaplin M. M. Munson S. T. Knuth 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1997,167(3):197-203
The effect of various activity regimes on metabolism of pigeon pectoralis was examined by measurement of blood lactate following
exercise, total lactate dehydrogenase activity of pectoral muscle, and proportions of specific isoenzymes of pectoral muscle
lactate dehydrogenase. Sprint-trained birds had the highest pectoral muscle lactate dehydrogenase activity (1409 IU · g−1 wet tissue), while endurance-trained birds had the highest peak lactate levels (287 mg · dl−1, extrapolated from decay curves) and fastest half-time of the lactate response (4.8 min) following exercise, but the lowest
lactate dehydrogenase activity (115 IU · g−1 wet tissue). Immobilization of one wing for 3 weeks following endurance training produced a marked increase in lactate dehydrogenase
activity of the immobilized muscle, compared to that in the contralateral pectoralis and endurance-trained muscle. Aerobic
forms of the lactate dehydrogenase enzyme (that favor conversion of lactate to pyruvate) predominated in pectoral muscle of
endurance-trained birds, while cage-confined birds exhibited primarily the anaerobic isoenzymes. These results demonstrate
that conversion of pectoral muscle lactate dehydrogenase isoenzymes, total lactate dehydrogenase activity, and half-time of
lactate response after exercise is dependent on activity regime in pigeons. In this respect, pigeon pectoral muscle responds
to training and disuse in a manner similar to that of mammalian skeletal muscle.
Accepted: 10 September 1996 相似文献
10.
We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions. 相似文献
11.
Rabbit muscle troponin complex covalently bound to CNBr-activated Sepharose 4B was shown to interact with soluble lactate dehydrogenase with a stoichiometry of 2 mol lactate dehydrogenase/mol of troponin. The presence of Ca2+ influenced the strength of association (the KD values of 0.73 and 2.3 microM were determined in the presence of 200 microM EGTA or 100 microM Ca2+, respectively). In the absence of Ca2+, the affinity of lactate dehydrogenase to troponin was strongly pH-dependent, reaching a maximum in the region of pH 6.0-7.0. No change of catalytic activity was observed as a result of interaction between lactate dehydrogenase and troponin, the enzyme appeared capable of functioning in the bound form. 相似文献
12.
The temperature stability of the cytoplasmic enzyme of the glycolysis of lactate dehydrogenase from a pig muscle (isoenzyme M4) in a complex with the anion polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, the own protein fluorescence, and circular dichroism. Calorimetric investigations of complex of lactate dehydrogenase with poly(styrenesulphonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulphonate)/lactate dehydrogenase, though at 20 degrees C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulphonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte. 相似文献
13.
O G Grebenshchikova L E Andrianova V N Prozorovski? 《Biokhimii?a (Moscow, Russia)》1989,54(3):361-369
Solid phase immunoenzymatic analysis was used to study the antigenic activity of proteolytic degradation products of the porcine muscle lactate dehydrogenase isoform M4. The presence in the enzyme structure of topographic (linear) antigenic determinants was demonstrated. Peptide 180-214 containing histidine-195 in the active center of lactate dehydrogenase was isolated from the tryptic hydrolysate of the carboxymethylated enzyme. This peptide interacts with antibodies against the native enzyme, i.e., antibodies bound to the immunoadsorbent, and causes a 20-25% inhibition of the antigen-antibody complex formation. Protein modification by fluorescein mercuriacetate at Cys-165 essential for the enzyme activity does not result in the synthesis of antibodies that would stimulate the inhibition of the lactate dehydrogenase catalytic activity as compared to antibodies to the native isoenzyme. The putative role of some amino acid residues in the structure of antigenic determinants of porcine muscle lactate dehydrogenase is discussed. 相似文献
14.
N P Sugrobova T B Eronina N A Chebotareva M V Ostrovskaia N B Livanova 《Molekuliarnaia biologiia》1983,17(2):430-436
The binding of pig skeletal muscle lactate dehydrogenase by F-actin has been studied using the sedimentation method in 10 mM Tris-acetate buffer, pH 6.0 at 20 degrees C. Adsorption capacity of F-actin is equal to (1 +/- 0.1) . 10(-5) moles of lactate dehydrogenase per 1 g of actin. NADH decreases the affinity of F-actin with respect to lactate dehydrogenase. The binding of lactate dehydrogenase by F-actin in diminishing the rate of enzymatic reduction of alpha-ketoglutarate. The microscopic dissociation constant for the complex of the enzyme with F-actin which is estimated from the dependence of the enzymatic reaction rate of F-actin concentration at saturating NADH concentrations is equal (3.0 +2- 0.5) . 10(-7) M. It has been shown that the bound enzyme is characterized by the greater value of Km and the lower value of Vmax in comparison to the free enzyme. 相似文献
15.
E. V. Durdenko S. M. Kuznetsova S. A. Tikhonenko V. I. Emelyanenko E. A. Saburova 《Biophysics》2010,55(4):535-543
The temperature stability of the cytoplasmic enzyme of glycolysis, lactate dehydrogenase from pig muscle (isoenzyme M4) in
complex with anionic polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential
scanning microcalorimetry, own protein fluorescence, and circular dichroism. Calorimetric investigations of the complex of
lactate dehydrogenase with poly(styrenesulfonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the
transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulfonate)/lactate
dehydrogenase, though at 20°C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective
of the addition of poly(styrenesulfonate). The addition of phosphate ions to the solution enhances the resistance of lactate
dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the
viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance
its resistance to both thermal denaturation and destruction by polyelectrolyte. 相似文献
16.
The changes in conformation of F-actin induced by the binding of the glycolytic enzyme lactate dehydrogenase were studied in myosin-free single ghost muscle fibres. The formation of the lactate dehydrogenase-F-actin complex was accompanied by changes in the parameters of intrinsic (tryptophan) and extrinsic (rhodaminyl-phalloin) polarized fluorescence of ghost muscle fibre F-actin. Lactate dehydrogenase stimulated actin-activated Mg2+-ATPase of myosin subfragment 1 by 30%. F-actin of ghost fibres depressed lactate dehydrogenase activity to 20% of the initial values. It is suggested that the energy-providing mechanism is coupled with that of muscle contraction through conformational changes in F-actin. 相似文献
17.
Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.Hu Lin is one of the cooperator of the experiment. 相似文献
18.
The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
The catalytic properties of the purified horseshoe crab and seaworm d-lactate dehydrogenases were determined and compared with those of several l-lactate dehydrogenases. Apparent Km's and degrees of substrate inhibition have been determined for both enzymes for pyruvate, d-lactate, NAD+ and NADH. They are similar to those found for l-lactate dehydrogenases. The Limulus “muscle”-type lactate dehydrogenase is notably different from the “heart”-type lactate dehydrogenase of this organism in a number of properties.The Limulus heart and muscle enzymes have been shown by several criteria to be stereospecific for d-lactate. They also stereospecifically transfer the 4-α hydrogen of NADH to pyruvate. The turnover number for purified Limulus muscle lactate dehydrogenase is 38,000 moles NADH oxidized per mole of enzyme, per minute. Limulus and Nereis lactate dehydrogenases are inhibited by oxamate and the reduced NAD-pyruvate adduct.Limulus muscle lactate dehydrogenase is stoichiometrically inhibited by para-hydroxymercuribenzoate. Extrapolation to two moles parahydroxymercuribenzoate bound to one mole of enzyme yields 100% inhibition. Alkylation by iodoacetamide or iodoacetate occurs even in the absence of urea or guanidine-HCl. Evidence suggests that the reactive sulfhydryl group may not be located at the coenzyme binding site.Reduced coenzyme (NADH or the 3-acetyl-pyridine analogue of NADH) stoichiometrically binds to Limulus muscle lactate dehydrogenase (two moles per mole of enzyme).Several pieces of physical and catalytic evidence suggest that the d- and l-lactate dehydrogenase are products of homologous genes. A consideration of a possible “active site” shows that as few as one or two key conservative amino acid changes could lead to a reversal of the lactate stereospecificity. 相似文献
20.
A S Levi 《Archives of biochemistry and biophysics》1975,168(1):115-121
Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower Km values for its substrates. Km values for pyruvate and lactate were 8 × 10 ?5m and 4 × 10?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H4) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M4) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules. 相似文献