scholarly journals Specific alkylation of a histidine residue in carnitine acetyltransferase by bromoacetyl-l-carnitine

1970 ◽  
Vol 116 (4) ◽  
pp. 713-720 ◽  
Author(s):  
J. F. A. Chase ◽  
P. K. Tubbs
Keyword(s):  
Enzyme Activity ◽  
Alkylation Reaction ◽  
Histidine Residue ◽  
Carnitine Acetyltransferase ◽  
Irreversible Loss ◽  
Total Acid ◽  
Active Centre ◽  
Low Concentrations ◽  
Spontaneous Hydrolysis ◽  
Hydrolysis Of

Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[14C]bromoacetyl-l-carnitine yields N-3-carboxy[14C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue.

scholarly journals Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations

1967 ◽  
Vol 102 (3) ◽  
pp. 917-921 ◽  
Author(s):  
R.H. Eaton ◽  
D. W. Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Competitive Inhibitor ◽  
Tissue Preparation ◽  
Intestinal Alkaline Phosphatase ◽  
Active Centre ◽  
Low Concentrations ◽  
Type 3 ◽  
Intestinal Preparation ◽  
Hydrolysis Of ◽  
Single Enzyme

1. Inhibition of the pyrophosphatase and orthophosphatase activities of human liver and small-intestinal alkaline-phosphatase preparations by different classes of inhibitors has been studied. 2. Each type of substrate, pyrophosphate or orthophosphate, is a competitive inhibitor of hydrolysis of the other type. 3. l-Phenylalanine is a non-competitive inhibitor of both types of activity of the intestinal preparation, but inhibits neither activity of the liver enzyme. Arsenate is a competitive inhibitor of both activities of both preparations. For a given inhibitor, the values of K(i) are independent of the type of substrate used when measurements are made at the same pH. 4. Mg(2+) ions activate orthophosphatase but inhibit pyrophosphatase, except in very low concentrations. 5. These results are compatible with the presence in each tissue preparation of a single enzyme with one type of active centre, possessing both orthophosphatase and pyrophosphatase activities.

scholarly journals Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase

1990 ◽  
Vol 271 (3) ◽  
pp. 701-706 ◽  
Author(s):  
A J Barrett ◽  
M A Brown
Keyword(s):  
Metal Ions ◽  
Chicken Liver ◽  
Time Dependent ◽  
Histidine Residue ◽  
Diethyl Pyrocarbonate ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Related Compounds ◽  
Single Enzyme

Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.

Regulation of cellulase from Ruminococcus

1972 ◽  
Vol 18 (3) ◽  
pp. 347-353 ◽  
Author(s):  
M. C. Fusee ◽  
J. M. Leatherwood
Keyword(s):  
Enzyme Activity ◽  
Cell Growth ◽  
Catabolite Repression ◽  
Specific Activity ◽  
Product Inhibition ◽  
Enzyme Synthesis ◽  
Energy Sources ◽  
Low Concentrations ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

The regulation of cellulase was examined in Ruminococcus albus and R. flavefaciens. Hydrolysis of cellulose, as shown by the formation of clear zones around the colonies of bacteria grown in cellulose-agar roll tubes, was inhibited by moderate levels of cellobiose. An intermediate in the metabolism of cellobiose may be responsible for the inhibition since strains which can use either sucrose or lactose were similarly inhibited by these energy sources. The inhibition of cellulase was examined in relation to either repression of enzyme synthesis or product inhibition of the enzyme activity. There was no inhibition by cellobiose added either to the routine enzymatic assay or to assays using low concentrations of carboxymethylcellulose. A repression mechanism was indicated by the decrease in specific activity of cultures grown in higher concentrations of cellobiose. The specific activity was calculated as the enzymatic activity on carboxymethylcellulose with respect to cell growth. The mechanism of repression was not distinguished between the model proposed by Jacob and Monod and catabolite repression. The growth of R. albus cultured in cellobiose–cellulose liquid medium exhibited a diauxic pattern similar to that described by Monod.

β-Lactamase inactivation by mechanism-based reagents

1980 ◽  
Vol 289 (1036) ◽  
pp. 309-319 ◽  
Keyword(s):  
Enzyme Activity ◽  
Mode Of Action ◽  
Structural Features ◽  
Irreversible Loss ◽  
E Coli ◽  
Mechanistic Pathway ◽  
Hydrolysis Of ◽  
Derivatives Of ◽  
Enzyme Intermediate ◽  
Acyl Derivatives

The mechanistic pathway followed by the E. coli R TEM ß-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the ß-lactamase-catalysed hydrolysis of the 7-a-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known ß-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a ß-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N -acyl derivatives of 6-ß-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the ß-lactamase.

scholarly journals Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.

1981 ◽  
Vol 154 (3) ◽  
pp. 856-867 ◽  
Author(s):  
M K Pangburn ◽  
R D Schreiber ◽  
H J Müller-Eberhard
Keyword(s):  
Hemolytic Activity ◽  
Alternative Pathway ◽  
Sulfhydryl Group ◽  
Fluid Phase ◽  
Factor H ◽  
Low Concentrations ◽  
Spontaneous Hydrolysis ◽  
Thioester Bond ◽  
Fragmentation Patterns ◽  
Hydrolysis Of

Activation of the alternative pathway of complement commences with the formation of an initial fluid-phase C3 convertase. Treatment of C3 with the nucleophilic reagent methylamine has previously been shown to result in the cleavage of an intramolecular thioester bond and to induce C3b-like properties, including the ability to form a fluid-phase C3 convertase. This report examines the hypothesis that spontaneous hydrolysis of the thioester generates a derivative of C3 that is responsible for the formation of the initial C3 convertase of the alternative pathway. The rate of spontaneous decay of C3 hemolytic activity in buffer was found to be between 0.2 and 0.4%/h. In the presence of other alternative pathway proteins, the rate of inactivation was 1%/h. The rate of spontaneous inactivation was greatly accelerated by low concentrations of chaotrophic agents such as KSCN or guanidine. Liberation of a sulfhydryl group, not present in native C3, correlated with loss of hemolytic activity, indicating that exposure to chaotropic agents resulted in thioester hydrolysis. Unlike native C3, C3 bearing a single reactive sulfhydryl group was capable of generating fluid-phase C3 convertase with Factors B, D, and P and was cleaved by Factor I (C3b inactivator) in the presence of Factor H (beta 1H). The fragmentation patterns indicated that the C3a domain was covalently associated with the functionally C3b-like C3. Organomercurial agarose was employed for the rapid removal of sulfhydryl-bearing, hemolytically inactive forms of C3 and C3b from native hemolytically active C3.

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

THE HYDROLYSIS OF MONOPHOSPHOINOSITIDE BY EXTRACTS OF BRAIN

1967 ◽  
Vol 45 (6) ◽  
pp. 853-861 ◽  
Author(s):  
W. Thompson
Keyword(s):  
Fatty Acids ◽  
Enzyme Activity ◽  
Calcium Ions ◽  
Brain Extract ◽  
Monovalent Cations ◽  
High Concentration ◽  
Diffusible Factor ◽  
Hydrolysis Of ◽  
The Brain ◽  
Long Chain

The hydrolysis of monophosphoinositide by soluble extracts from rat brain is described. Diglyceride and inositol monophosphate are liberated along with a small amount of free fatty acids. Hydrolysis of the lipid is optimal at pH 5.4 in acetate buffer. The reaction is stimulated by calcium ions or by high concentration of monovalent cations and, to a less extent, by long-chain cationic amphipathic compounds. Enzyme activity is lost on dialysis of the brain extract and can be restored by diffusible factor(s). Some differences in the conditions for hydrolysis of mono- and tri-phosphoinositides are noted.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

scholarly journals Induction of carnitine acetyltransferase by clofibrate in rat liver

1981 ◽  
Vol 194 (1) ◽  
pp. 249-255 ◽  
Author(s):  
B Mittal ◽  
C K R Kurup
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Time Lag ◽  
Mitochondrial Protein ◽  
Carnitine Acetyltransferase ◽  
Enzyme Protein ◽  
General Protein Synthesis ◽  
Liver And Kidney ◽  
General Protein

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

LOCALIZATION AND CHARACTERIZATION OF CHOLINESTERASE IN SUBCELLULAR FRACTIONS OF RAT BRAIN AND BEEF PITUITARY

1961 ◽  
Vol 39 (9) ◽  
pp. 1335-1345 ◽  
Author(s):  
Surendra S. Parmar ◽  
Morley C. Sutter ◽  
Mark Nickerson
Keyword(s):  
Rat Brain ◽  
Cholinesterase Activity ◽  
Succinic Dehydrogenase ◽  
Anterior Lobe ◽  
Subcellular Fractions ◽  
Posterior Pituitary ◽  
Low Concentrations ◽  
Pituitary Glands ◽  
Hydrolysis Of

Fresh rat brains and fresh anterior and posterior pituitary glands of beef were separated by differential centrifugation into subcellular fractions, characterized on the basis of sedimentation and succinic dehydrogenase activity. Cholinesterase activity was measured by both manometric and colorimetric methods, the results of which were comparable. Cholinesterase activity of rat brain was found mainly in the microsome and supernatant fractions. It was quite uniformly distributed in all subcellular fractions of both anterior and posterior pituitary. Comparisons of the relative rates of hydrolysis of acetylthiocholine and butyrylthiocholine, and of inhibition by eserine, indicated that brain contains a much higher percentage of acetylcholinesterase than do both lobes of the pituitary, which contain relatively low concentrations of the specific enzyme. Total cholinesterase activity and its sensitivity to inhibition by eserine in the posterior pituitary were found to be midway between those of the anterior lobe and of the brain, from which the posterior pituitary was derived during embryological development.

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