Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human sperm chromosomes can be visualized after fusion with hamster eggs. Most laboratories use one of two methods of sperm treatment for capacitation: incubation in a modified Krebs-Ringer medium (BWW) for 5-7 h at 37 degrees C or storage in a TES-Tris yolk buffer (TYB) for 24-72 h at 4 degrees C. To determine whether data from the two methods were comparable, we performed a series of controlled experiments on one normal donor in which ejaculates were split and one aliquot of sperm was capacitated in BWW for 5-7 h at 37 degrees C (fresh) and the second aliquot was capacitated in TYB for 48 h at 4 degrees C (TYB). After capacitation, the technique used to obtain human sperm chromosome complements was identical for both aliquots. Both fresh and TYB sperm were further subdivided into two groups, which were subjected to either a short (1 h) or a long (3 h) gamete coincubation in BWW. This experiment was performed to determine if the longer incubation in BWW might induce chromosomal fragile sites and breaks because of nutritional depletion of the medium. A total of 458 human sperm chromosome complements was analysed. There was no significant difference in the frequency of sperm chromosomal abnormalities or in the sex ratio in the sperm coincubated with eggs for a short (1 h) or long (3 h) time in BWW. When sperm pretreatments were compared, there was a significant increase in the frequency of total sperm chromosomal abnormalities after TYB storage compared to fresh treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1990 Jun
PMID:Effect of culture conditions and media on the frequency of chromosomal abnormalities in human sperm chromosome complements. 237 92

Urine with trace amounts of different proteins from healthy people or B-lymphoma patients was concentrated and separated simultaneously by counterflow isotachophoresis on cellulose acetate membranes (CAM). The protein zones were blotted onto nitrocellulose membrane (NCM) by direct contact of CAM and NCM. NCM-blots were exposed to second isotachophoresis with the leading electrolyte 0.06 M Tris-HCl and the terminating one, 0.012 M Tris-beta-alanine. Under these conditions the moving boundary formed by Cl-/beta-alanine- migrated towards the anode with decreasing velocity. At a certain point the rate of migration of the moving boundary became completely compensated by the electroendosmotic counterflow. In this steady state position the boundary stopped on the NCM support, while the electroendosmotic rate in the area before the boundary was much higher than the rate of the opposite migration of any protein to the anode. Under these conditions electroendosmosis served as a "conveyer belt" which transferred consecutively the immunoreagents, antibodies, immunoconjugates, or antiperoxidase-peroxidase system through the protein blots "printed" on NCM. The immunoblots obtained in this way were developed by the substrate for the immunoenzyme complex used in the experiment. The technique could be used to characterize light chains present in the urine of normal donors and monoclonal light chains in the urine of patients with B-cell malignancies.
Mol Immunol 1989 Jan
PMID:Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes--I. Immunoblotting. 249 35

A calcium-sensitive, lipid-dependent protein kinase (protein kinase C) modulates physiological function in a variety of cells. In certain tissues, multiple forms of this key regulatory enzyme exist. To examine the nature of ovarian protein kinase C, swine luteal cytosol (1000 mg protein) was applied and eluted from DE-52 cellulose. The pooled active fractions were sequentially purified further by threonine- (TS) and phenyl-Sepharose (PS) affinity chromatography in a buffer of 20 mM Tris, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM dithiothreitol and 250 micrograms/l leupeptin. Protein kinase C activity was eluted with NaCl gradients of 0.075-125 M (DE-52), 0.1-1 M (TS) and 0.6-0.0 M (PS). This purification scheme yielded three distinct peaks of highly purified protein kinase C activity and an overall enrichment in protein kinase C activity of approximately 1000-fold. Inasmuch as control of steroidogenesis or peptide hormone production in the ovary via the Ca2+-protein kinase C pathway could occur at several different levels, we postulate that the demonstrated presence of isoforms of this enzyme in ovarian tissue offers a means for selective spatial and temporal compartmentalization of the endocrine stimulus with correspondingly distinct changes in cellular function.
Mol Cell Endocrinol 1989 Jan
PMID:Purification of three forms of chromatographically distinct protein kinase C from the swine ovary. 250 Nov 18

We report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E. coli chromosome as well as a number of single-stranded bacteriophages. The procedure starts with E. coli cells harboring an overproducing plasmid, pRepO, in which the E. coli rep gene is under transcriptional control of the inducible lambda PL promoter (Colasanti, J., and Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in greater than 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells. We also show that cell death occurs upon inducing such a large overproduction of the E. coli Rep protein in MZ-1/pRepO. The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality. The extinction coefficient of purified E. coli Rep protein is epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees C. The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37 degrees C (pH 7.5). Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility. At 4 degrees C, in the presence of 20% glycerol and greater than or equal to 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (+/- ATP and +/- MgCl2). The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has approximately 40% of its activity as compared to solutions that contain no glycerol. The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate greater than acetate greater than chloride. The ability to obtain highly purified E. coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.
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PMID:Large-scale purification and characterization of the Escherichia coli rep gene product. 252 89

The effects of acidosis (pH 6.5) on the efflux of noradrenaline from the perfused heart of the rat have been studied. Acidosis does not influence the noradrenaline efflux induced by sympathetic nerve stimulation either in the presence or absence of neuronal uptake blockade. It is therefore unlikely that acidosis will contribute to the failure of nerve stimulation mediated noradrenaline release previously shown to occur during myocardial ischaemia. Acidosis exerts a biphasic effect on the noradrenaline efflux produced by substrate free anoxic perfusion with an inhibition of early noradrenaline overflow. Peak anoxic efflux of noradrenaline is greater when extracellular NaCl is replaced by Tris or sucrose in keeping with the hypothesis of carrier-mediated noradrenaline efflux. Marked early anoxic efflux occurs when extracellular NaCl is replaced by LiCl suggesting an additional mechanism of vesicular destabilization. Anoxic noradrenaline efflux is inhibited by amiloride (and ethylisopropyl amiloride) and it is proposed that the inhibitory effect of extracellular acidosis on early noradrenaline efflux also occurs by inhibition of the Na+ (Li+)/H+ antiporter leading to reduced Na+ (Li+) entry during the early phase of anoxia. At a later stage acidosis enhances anoxic noradrenaline efflux. This effect is postulated to be due to a reduction in the transvesicular pH gradient available for catecholamine storage within the storage vesicles.
J Mol Cell Cardiol 1989 Jan
PMID:Effects of acidosis on anoxic and exocytotic noradrenaline release from the heart. 254 Dec 58

In low ionic strength buffer (5 mM Tris.HCl), the binding of [3H] nitrendipine to dihydropyridine calcium antagonist binding sites of mouse forebrain membranes is increased by both Na+ and Ca2+. Radiation inactivation was used to determine the target size of [3H]nitrendipine binding sites in 5 mM Tris.HCl buffer, in the presence and absence of these cations. After irradiation, [3H] nitrendipine binding in buffer with or without Na+ was diminished, due to a loss of binding sites and also to an increase in Kd. After accounting for radiation effects on the dissociation constant, the target size for the nitrendipine binding site in buffer was 160-170 kDa and was 170-180 kDa in the presence of sodium. In the presence of calcium ions, [3H]nitrendipine binding showed no radiation effects on Kd and yielded a target size of 150-170 kDa. These findings suggest, as in the case of opioid receptors, the presence of high molecular weight membrane components that modulate cation-induced alterations in radioligand binding to dihydropyridine binding sites.
Mol Pharmacol 1989 Aug
PMID:Radiation inactivation reveals discrete cation binding sites that modulate dihydropyridine binding sites. 254 88

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was the only reaction substrate which provided protection from inactivation. Acetyl-CoA did not affect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and the presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (k) were 0.11 and 0.26 min-1, respectively. oATP completely inhibited the reaction of [14C]ADP in equilibrium ATP exchange, whereas produced actually no effect on [14C]acetyl-CoA equilibrium with malonyl-CoA exchange. Incorporation of about one equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No restoration of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB[3H]4 has not led to 3H incorporation. The modification process involves elimination of the triphosphate chain of oATP. The results obtained indicate the affinity character of oATP-mediated modification of acetyl-CoA carboxylase. The reagent apparently interacts selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.
Mol Biol (Mosk)
PMID:[Acetyl-CoA-carboxylase: modification of ATP-binding site of the active center by 2',3'-dialdehyde derivative of ATP]. 257 82

Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37 degrees C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K+), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH4Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37 degrees C, but not at 0 degrees C. The Mg++-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl2 in the presence of 1mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H+ ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.
Mol Cell Biochem 1989 Oct 31
PMID:Is the ATP-dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump. 258 96

Casein kinase II from a virally-transformed macrophage cell line (RAW264) was purified by a sequential DEAE, Procion Red, phosvitin-Sepharose and heparin-Sepharose chromatography. With [tau-32P]GTP as a phosphate donor and casein as a substrate, the kinase was stimulated by polyamines and inhibited by heparin. The purified kinase had a specific activity of 1137 nmol/min/mg protein and exhibited three major protein bands of 40 K, 35 K, and 25 K. Under non-denaturing conditions in 50 mM Tris-50 mM NaCl the enzyme was eluted as a single peak with molecular weight of 110 K. Incubation of kinase in the presence of [tau-32P]GTP and Mg2+ resulted in phosphorylation of the 25 K protein band of the enzyme. In the presence of [tau-32P]GTP and Mg2+ the kinase was able to phosphorylate 55 K protein band in purified ornithine decarboxylase preparation from RAW264 cells and the rat-type II regulatory subunit of the cyclic AMP-dependent protein kinase.
Cell Mol Biol 1989
PMID:Characterization of casein kinase II from a virally transformed macrophage-like cell line, RAW264. 262 1

The specific DNA-binding protein FIS (factor for inversion stimulation), which stimulates site-specific DNA inversion by interaction with an enhancer sequence, was purified from an Escherichia coli strain overproducing the protein. FIS was crystallized at room temperature by microdialysis against 1.2 to 1.5 M-sodium/potassium phosphate containing 10 mM-Tris.HCl, 0.5 to 1 M-NaCl and 1 mM-NaN3 at pH 8.0 to 8.2. The crystals are stout prisms and suitable for X-ray diffraction study beyond 2.5 A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1). The unit cell has dimensions a = 47.57(4) A, b = 51.13(4) A, c = 79.83(6) A and contains one FIS dimer in the asymmetric unit.
J Mol Biol 1989 Jul 05
PMID:Crystallization of the DNA-binding Escherichia coli protein FIS. 267 86


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