The interaction of rhodium(II) carboxylates with enzymes.
(1/1153)
The effect of rhodium(II) acetate, propionate, and methoxyacetate on the activity of 17 enzymes was evaluated. The enzymes were preincubated with the rhodium(II) complexes in order to detect irreversible inhibition. All enzymes that have essential sulfhydryl groups in or near their active site were found to be irreversibly inhibited. Those enzymes without essential sulfhydryl groups were not affected. In each case, the rate of inactivation closely paralleled the observed toxicity and antitumor activity of rhodium(II) carboxylates; that is, rhodium(II) propionate greater than rhodium(II) acetate greater than rhodium(II) methoxyacetate. In addition, those enzymes that have been demonstrated to be most sensitive to established sulfhydryl inhibitors, such as glyceraldehyde-3-phosphate dehydrogenase, were also most sensitive to rhodium(II) carboxylate inactivation. Proton nuclear magnetic resonance measurements made during the titration of rhodium(II) acetate with cysteine showed that breakdown of the carboxylate cage occurred as a result of reaction with this sulfhydryl-containing amino acid. (+info)
Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor.
(2/1153)
A radio-ribonuclease inhibitor assay based on the interaction of 125I-angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic-type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly-arginine, poly-lysine and poly-ornithine, core histones, spermatid-specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly-arginine and poly-lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly-lysine and protamines while 125I-angiogenin did not. Antagonists of the angiogenin-RI interaction are potential regulators of either angiogenin-triggered angiogenesis and/or intracellular RI function, depending on their preferential target. (+info)
Destruction of protamine in human sperm inhibits sperm binding and penetration in the zona-free hamster penetration test but increases sperm head decondensation and male pronuclear formation in the hamster-ICSI assay.
(3/1153)
PURPOSE: Our purpose was to investigate the fertilizing ability of human protamine-damaged sperm in a heterologous system using hamster oocytes. METHODS: The protamine of the sperm were damaged by exposure to dithiothreitol, a disulfide-reducing agent. Their ability to penetrate and form male pronuclei were investigated using the zona-free hamster penetration test and the hamster-intracytoplasmic sperm injection assay, respectively. RESULTS: The zona-free hamster penetration test revealed that protamine-damaged sperm are unable to bind and penetrate the hamster oocyte. On the other hand, hamster-intracytoplasmic sperm injection assay results showed that 56.9% and 39.2% of the injected oocytes developed male pronuclei in protamine-damaged and live-intact sperm groups, respectively, with a significant difference in these rates (P < 0.01). CONCLUSIONS: This study shows that protamine-damaged sperm are able to undergo sperm head decondensation and male pronuclear formation only when injected into the ooplasm, although they cannot bind and penetrate through the zona and enter the ooplasm. (+info)
The covalent attachment of polyamines to proteins in plant mitochondria.
(4/1153)
Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases. (+info)
Preparation and properties of S-cyano derivatives of creatine kinase.
(5/1153)
The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5'-dithio-bis(2-nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol-blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8 - 0.9-SH group per subunit. This mono-S-cyano derivative of creatine kinase was found 73% as active as the native unmodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted in the bis-S-cyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme. (+info)
Solubilization of diglyceride acyltransferase from the membrane of Mycobacterium smegmatis.
(6/1153)
Diglyceride acyltransferase [acyl-CoA : 1,2-diacylglycerol O-acyltransferase, EC 2.3.1.20] was found to be localized in the membrane of Mycobacterium smegmatis, and this enzyme could be solubilized from the membrane by treatment with aqueous acetone. The solubilized enzyme required either 1,2-diolein or 1, 3-diolein as an acceptor for palmitoyl-CoA. The apparent Km value for 1,2- or 1,3-diolein and that for palmitoyl-CoA were about 1.4 X 10(-5) M and 6 X 10(-6) M, respectively. Several sulfhydryl reagents were inhibitory to the enzyme activity, suggesting the existence of a thiol group(s) in its active site. The solubilized enzyme, which was more labile than that membrane-bound one, could be stabilized to some extent with antichaotropic salts such as phosphate, pyrophosphate, and sulfate. (+info)
The aconitase of yeast. IV. Studies on iron and sulfur in yeast aconitase.
(7/1153)
Chemical analyses were carried out to determine the active components of the crystalline aconitase [EC 4.2.1.3] of Candida lipolytica. The enzyme contained 2 atoms of non-heme iron, 1 atom of labile sulfur, and 6 sulfhydryl groups per molecule. One atom of the non-heme iron was released by the addition of metal-chelating agents such as sodium citrate, sodium nitrilotriacetate (NTA) or sodium ethylenediaminetetraacetate (EDTA) without loss of the enzyme activity. The non-heme iron and labile sulfur were released by the addition of sulfhydryl reagents such as rho-chloromercuribenzoate (PCMB), sodium mersalyl or urea with loss of the enzyme activity. o-Phenanthroline reacted with the iron atoms in the enzyme at pH 6.0 with loss of the activity. These results show that yeast aconitase is an iron-sulfur protein and that only one of the two non-heme iron atoms is essential for enzyme activity. (+info)
Effect of diluent temperature on creatine kinase values found for lyophilized controls and reference sera.
(8/1153)
We report the effect of temperature of diluent on creatine kinase activity in several lyophilized controls. Creatine kinase activity was significantly greater when the lyophilized control was reconstituted with diluent at 4 degrees C as compared to 25 degrees C. This is an additional source of variation in creatine kinase controls. (+info)