STUDIES ON THE CONFORMATIONAL CHANGES OF MITOCHONDRIAL MALATE DEHYDROGENASE IN UREA–PHOSPHATE SOLUTIONS

Pig-heart mitochondrial malate dehydrogenase is gradually inactivated in 4 M urea. During the inactivation, sulfhydryl groups on the protein are exposed in a first-order reaction. The reaction is followed spectrophotometricaily using the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). Titration with DTNB in the presence of urea exposes 10 to 12 sulfhydryl groups per molecule of mitochondrial malate dehydrogenase. The enzyme is also inactivated when diluted in water but no sulfhydryl groups are unmasked. The loss of activity and the appearance of sulfhydryl groups in urea solutions do not take place at the same rate.The conformational changes of malate dehydrogenase that occur in urea solutions are partially prevented by inorganic phosphate ions, and less so by the substrates NADH, NAD+, oxalacetate (OAA), and L-malate. The protection against loss of enzyme activity by inorganic phosphate ions is pH-dependent. Both inorganic phosphate and NADH considerably reduce the first-order rate constant for sulfhydryl appearance in 4 M urea. Protection of the enzyme against sulfhydryl appearance in urea solutions by pre-incubation with the substrates indicates that about two sulfhydryl groups per molecule of mitochondrial malate dehydrogenase are involved in substrate binding. Thus, the substrates must keep the active site of the enzyme intact. They either bind to the sulfhydryl groups or prevent the protein molecule from completely unfolding.

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Iodoacetate inactivation of rape alcohol dehydrogenase

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19801601 ◽

1980 ◽

Vol 45 (5) ◽

pp. 1601-1607 ◽

Cited By ~ 1

Author(s):

Marie Stiborová ◽

Sylva Leblová

Keyword(s):

Alcohol Dehydrogenase ◽

Protein Molecule ◽

Sulfhydryl Groups ◽

Inactivation Rate ◽

First Order ◽

Ph Dependent ◽

Kinetics Of

Iodoacetate inactivates rape alcohol dehydrogenase (ADH, EC 1.1.1.1). The inactivation rate follows the kinetics of the first order, is pH-dependent, and decreases below pH 7.5. Besides irreversible alkylation of the sulfhydryl groups of the enzyme iodoacetate also forms a reversible complex with rape ADH. The coenzyme (NAD) and its analogs (ATP, ADP, AMP) competitively protect the enzyme against alkylation; o-phenanthroline also protects the enzyme against alkylation yet noncompetitively with respect to iodoacetate. Imidazole and o-phenanthroline compete with one another for binding to the protein molecule of rape ADH. Whereas o-phenanthroline decreases the inactivation rate imidazole increases the rate of iodoacetate inactivation.

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Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility

Biochemical Journal ◽

10.1042/bj2210289 ◽

1984 ◽

Vol 221 (2) ◽

pp. 289-296 ◽

Cited By ~ 6

Author(s):

S C J Cole ◽

R J Yon

Keyword(s):

Conformational Changes ◽

Wheat Germ ◽

Binding Pocket ◽

Order Rate Constant ◽

Aspartate Transcarbamoylase ◽

Carbamoyl Phosphate ◽

Peptide Bonds ◽

First Order ◽

Mediated Effects ◽

Arginine Residues

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.

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Plasma clearance and endocytosis of mitochondrial malate dehydrogenase in the rat

Biochemical Journal ◽

10.1042/bj2000115 ◽

1981 ◽

Vol 200 (1) ◽

pp. 115-121 ◽

Cited By ~ 4

Author(s):

M K Bijsterbosch ◽

A M Duursma ◽

J M W Bouma ◽

M Gruber ◽

P Nieuwenhuis

Keyword(s):

Bone Marrow ◽

Enzyme Activity ◽

Malate Dehydrogenase ◽

Plasma Clearance ◽

Reticuloendothelial System ◽

First Order ◽

First Order Kinetics ◽

Long Run ◽

Mitochondrial Malate Dehydrogenase ◽

Adsorptive Endocytosis

1. Pig mitochondrial malate dehydrogenase was labelled with 125I and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of only 7 min. 2. Radioactivity accumulated in liver, spleen, bone (marrow) and kidneys, reaching maxima of 3 1, 4, 6 and 9% of the injected dose respectively, at 10 min after injection. 3. Our data allow us to calculate that in the long run 59, 5, 11 and 13% of the injected dose is taken up and subsequently broken down by liver, spleen, bone and kidneys respectively. 4. Differential fractionation of liver showed that the acid-precipitable radioactivity was mainly present in the lysosomal and microsomal fractions, suggesting that the endocytosed protein is transported via endosomes to lysosomes, where it is degraded. 5. Radioautography of liver and spleen suggested that the labelled protein was taken up by macrophages of the reticuloendothelial system. 6. Mitochondrial malate dehydrogenase is probably internalized in liver, spleen and bone marrow by adsorptive endocytosis, since uptake of the enzyme of these tissues is saturable.

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Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes

Biochemical Journal ◽

10.1042/bj1860227 ◽

1980 ◽

Vol 186 (1) ◽

pp. 227-233 ◽

Cited By ~ 5

Author(s):

K A Webster ◽

K B Freeman ◽

S Ohki

Keyword(s):

Malate Dehydrogenase ◽

Conformational Changes ◽

Ribonuclease A ◽

Phospholipid Vesicles ◽

Mitochondrial Matrix ◽

Dimer Formation ◽

Dimeric Form ◽

Oil Water ◽

Mitochondrial Malate Dehydrogenase

Porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) dissociates into subunits on dilution. The enzyme monomer caused large increases in the surface pressure of monolayers of 1:1 phosphatidylserine/phosphatidylcholine at air/water and oil/water interfaces. The monomer increased the permeability of phospholipid vesicles to 22Na+. Both effects were significantly greater than the corresponding effects of ribonuclease A, cytochrome c and the dimeric form of malate dehydrogenase. Changes in the circular-dichroism spectra of the enzyme indicated that conformational changes may be associated with dimer formation or when monomer interacts with lysophosphatidyl-choline. Similar interactions to those described may occur in situ when mitochondrial malate dehydrogenase is transported to the mitochondrial matrix from its site of synthesis on cytosolic ribosomes.

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Development of Sustained Release Multi-Component Microparticulate System of Diclofenac Sodium and Tizanidine Hydrochloride

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/10.37285/ijpsn.2008.1.1.9 ◽

1970 ◽

Vol 1 (1) ◽

pp. 97-105

Author(s):

Kamlesh Dashora ◽

Shailendra Saraf ◽

Swarnalata Saraf

Keyword(s):

Particle Size ◽

Sustained Release ◽

Cellulose Acetate ◽

Rate Constant ◽

Diclofenac Sodium ◽

Order Rate Constant ◽

Anomalous Transport ◽

First Order ◽

Sem Study ◽

Transport Type

Sustained released tablets of diclofenac sodium (DIC) and tizanidine hydrochloride (TIZ) were prepared by using different proportions of cellulose acetate (CA) as the retardant material. Nine formulations of tablets having different proportion of microparticles developed by varied proportions of polymer: drug ratio ‘’i.e.’’; 1:9 -1:3 for DIC and 1:1 – 3:1 for TIZ. Each tablet contained equivalent to 100 mg of DIC and 6mg of TIZ. The prepared microparticles were white, free flowing and spherical in shape (SEM study), with the particle size varying from 78.8±1.94 to 103.33±1.28 µm and 175.92± 9.82 to 194.94±14.28µm for DIC and TIZ, respectively. The first order rate constant K1 of formulations were found to be in the range of K1 = 0.117-0.272 and 0.083- 0.189 %hr-1for DIC and TIZ, respectively. The value of exponent coefficient (n) was found to be in the range of 0.6328-0.9412 and 0.8589-1.1954 for DIC and TIZ respectively indicates anomalous to non anomalous transport type of diffusions among different formulations

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