Properties of malate dehydrogenase isolated from Methanospirillum hungatii

1979 ◽  
Vol 25 (2) ◽  
pp. 192-200 ◽  
Author(s):  
G. D. Sprott ◽  
R. C. McKellar ◽  
K. M. Shaw ◽  
J. Giroux ◽  
W. G. Martin
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Reaction Mechanism ◽  
Malate Dehydrogenase ◽  
Initial Rate ◽  
Rate Data ◽  
Cell Extracts ◽  
Ping Pong ◽  
Sequential Mechanism ◽  
Variable Substrate

A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, α-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by α-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. α-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to α-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of initial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was preferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells with respect to molecular weight (61 700) and reaction mechanism, but may be regulated differently.

Ornithine transcarbamylase from Mycobacterium smegmatis ATCC 14468: purification, properties, and reaction mechanism

1986 ◽  
Vol 64 (12) ◽  
pp. 1349-1355 ◽  
Author(s):  
Suhail Ahmad ◽  
R. K. Bhatnagar ◽  
T. A. Venkitasubramanian
Keyword(s):  
Enzyme Activity ◽  
Reaction Mechanism ◽  
Mycobacterium Smegmatis ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Ornithine Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Michaelis Constants ◽  
Variable Substrate ◽  
Uncompetitive Inhibitor

Ornithine transcarbamylase (EC 2.1.3.3) has been purified 980-fold from Mycobacterium smegmatis and has a molecular weight of 116 000. Initial velocity determinations indicated that the reaction proceeds via a sequential kinetic mechanism. The limiting Michaelis constants for carbamyl phosphate (KmA) and ornithine (KmB) and the dissociation constant for carbamyl phospate (Kia) were found to be 0.20, 0.25, and 0.07 mM, respectively. Ornithine at higher concentrations acted as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Phosphate was a competitive inhibitor with carbamyl phosphate as variable substrate and showed noncompetitive or mixed type inhibition when ornithine was the variable substrate. Norvaline acted as a competitive inhibitor with ornithine as variable substrate and as an uncompetitive inhibitor when carbamyl phophate was the variable substrate. Such inhibitory patterns are characteristic of reactions that proceed via sequential ordered mechanisms. Although the enzyme activity was strongly inhibited by arginine, several arginine analogs had no effect on the enzyme activity. The results suggest that, even though the enzyme from M. smegmatis is unique in the sense that it is feedback inhibited by arginine, the reaction mechanism is similar to the ornithine transcarbamylase isolated from other microorganisms.

scholarly journals The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 139 (1) ◽  
pp. 27-35 ◽  
Author(s):  
W. H. Shaw ◽  
J. W. Anderson
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Exchange Reaction ◽  
Initial Rate ◽  
Leaf Tissue ◽  
Kinetic Studies ◽  
Forward Direction ◽  
Sequential Mechanism ◽  
Spinach Leaf ◽  
Atp Sulphurylase

1. Sulphate-dependent PPi–ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP2− and MgP2O72−; ATP sulphurylase activity was not correlated with the concentration of free Mg2+. 2. Sulphate-dependent PPi–ATP exchange was independent of PPi concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PPi–ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [32P]ATP from [32P]PPi and adenosine 5′-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PPi and adenosine 5′-sulphatophosphate. 5. The synthesis of ATP from PPi and adenosine 5′-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PPi and adenosine 5′-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5′-sulphatophosphate and non-competitive with respect to PPi. It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP2− was the first product to react with the enzyme and MgP2O72− was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5′-sulphatophosphate could not be demonstrated.

The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

1978 ◽  
Vol 40 (02) ◽  
pp. 350-357
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Molecular Weight ◽  
Dissociation Constant ◽  
Calcium Ions ◽  
Activation Process ◽  
Factor X ◽  
The Real ◽  
High Affinity ◽  
Binding Data ◽  
Sequential Mechanism ◽  
Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

The kinetics of hydration of tricalcium silicate

1970 ◽  
Vol 48 (21) ◽  
pp. 3291-3299 ◽  
Author(s):  
K. G. McCurdy ◽  
B. P. Erno
Keyword(s):  
Reaction Mechanism ◽  
Intermediate Product ◽  
Activation Energies ◽  
Tricalcium Silicate ◽  
Rate Data ◽  
Large Excess ◽  
First Order ◽  
Kinetics Of ◽  
Subsequent Decomposition

An investigation has been made of the kinetics of hydration of tricalcium silicate at several temperatures in a large excess of water in the presence of various added ions. The rate data have been interpreted by a reaction mechanism which involves: (a) the first order hydration of tricalcium silicate to form an intermediate product, 1.5CaO•SiO2, which can react by two pathways, (b) the direct first order decomposition of intermediate, 1.5CaO•SiO2, to form lime and silica or (b′) complexing of intermediate with silica and subsequent decomposition to form lime and silica. This reaction mechanism predicts the rate of production of base during the hydration. The effect of various added ions is interpreted in terms of the proposed mechanism.Rate constants and activation energies for the various steps in the proposed mechanism are reported.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

Enzymatic sulfation of steroids. V. Partial purification and some properties of sulfotransferase III, the major glucocorticoid sulfotransferase of liver cytosols from male rats

1978 ◽  
Vol 56 (11) ◽  
pp. 1028-1035 ◽  
Author(s):  
Sanford S. Singer ◽  
James Gebhart ◽  
Edward Hess
Keyword(s):  
Molecular Weight ◽  
Sulfhydryl Groups ◽  
Male Rats ◽  
Partial Purification ◽  
Ph Optimum ◽  
Fold Enrichment ◽  
Competitive Inhibitors ◽  
Sequential Mechanism ◽  
Inhibition Studies ◽  
Estradiol 17Β

This manuscript describes purification of sulfotransferase III (STIII), the major hepatic glucocorticoid sulfotransferase of male rats, 77.8 ± 16 fold from cytosol. This represents a probable 250–345 fold enrichment, compared with homogenates. Purified STIII has a molecular weight of 61 500 ± 2500 from Sephadex G-100 chromatography. It is markedly activated by 5 mM divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts; inhibited strongly by 5 mM divalent Zn and Cd; and unaffected by 8 mM ADP, ATP, and AMP. Comparison of the ability of purified STIII to sulfate equimolar Cortisol, estradiol-17β, testosterone, and dehydroepiandrosterone suggests that the enzyme may sulfate glucocorticoids preferentially. However, its Cortisol sulfotransferase activity is inhibited by a variety of steroids. Of these, dehydroepiandrosterone, dexamethasone, and progesterone were tested extensively. They were found to be competitive inhibitors. STIII has a sharp pH optimum at pH 6.0 ± 0.1. However, it is routinely assayed at pH 6.8, as explained in the text. It exhibits a sequential mechanism and Km values of 6.82 ± 1.2 and 6.28 ± 0.64 μM for Cortisol and 3′-phosphoadenosine-5′-phosphosulfate, respectively. It also possesses essential sulfhydryl groups, as shown by p-hydroxymercuribenzoate inhibition studies.

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