ovalbumin and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Emulsification of aqueous protein solutions in methylene chloride triggered the formation of water-insoluble aggregates at a water/methylene chloride interface. As a result, the amounts of beta-lactoglobulin and ovalbumin recovered in water were 36 and 44%, respectively. Addition of 5 mM: CHAPS in the aqueous phase raised the degree of beta-lactoglobulin recovery to 96%. Sodium taurocholate, however, failed to improve protein recovery. The stabilizing effect of CHAPS was also protein-specific and concentration-dependent: at >or=5 mM: , the surfactant caused unfolding of ovalbumin to make a water-soluble oligomer. CHAPS thus stabilizes proteins at an interface.

Topics: Cholic Acids; Dose-Response Relationship, Drug; Emulsifying Agents; Lactoglobulins; Methylene Blue; Ovalbumin; Proteins; Solubility; Surface-Active Agents; Taurocholic Acid

Using guanosine 5'-(3-O-thio)triphosphate (GTPgammaS), we previously reported that protein carboxyl methyltransferase activities in kidney brush border membranes were increased by the GTP analog (Arch. Biochem. Biophys. 351, 149-158, 1998). Here, we investigated the distribution and characterized the effect of GTPgammaS on protein carboxyl methylation activity. The analysis of species distribution of carboxyl methylation in kidney brush border membranes showed that the GTPgammaS strongly stimulated this activity in rat (15.9-fold), mouse (14.7-fold), human (2.9-fold), and rabbit (2.7-fold). Analysis of GTPgammaS-dependent carboxyl methylation in rat tissues and cell fractions indicated that the activity was mainly localized in membranes of intestine, lung, and kidney, with the highest activity found in liver. To characterize the methyltransferase activity modulated by GTPgammaS in liver membranes, their sensitivity to the detergent 3-[(3-cholamido)dimethylammonio]-1-propanesulfonic acid (Chaps) was used. Methylation of N-acetyl-S-farnesyl cysteine, a prenylated protein methyltransferase (PPMT) substrate was strongly inhibited (86%) in the presence of Chaps, while the methylation of bovine calmodulin and ovalbumin, both of which are substrates for the protein L-isoaspartyl/d-aspartyl methyltransferase (PIMT), was slightly reduced by the detergent (0-12%). The GTPgammaS-dependent carboxyl methylation of endogenous substrates in liver membranes was decreased by 35% in the presence of Chaps, suggesting that PPMT was not the predominant methyltransferase involved in the methylation stimulated by GTPgammaS in liver membranes. Electrophoretic analysis showed that radioactive methylation of several substrates induced by GTPgammaS in liver membranes was reduced by adding calmodulin. Interestingly, addition of GTPgammaS partially inhibited the methylation of two PIMT substrates, ovalbumin (24%) and bovine calmodulin (19%), when incubated with liver membranes. Immunoprecipitation of PIMT from liver and lung membranes strongly inhibited (88-94%) the methylation stimulated by GTPgammaS. Altogether, these data support the hypothesis that GTPgammaS could regulate PIMT activity and may provide new insights into the function of the methyltransferase.

Topics: Animals; Brain; Calmodulin; Cell Membrane; Cholic Acids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kidney; Liver; Male; Methylation; Mice; Ovalbumin; Protein D-Aspartate-L-Isoaspartate Methyltransferase; Protein Methyltransferases; Rabbits; Rats; Rats, Sprague-Dawley; Tissue Distribution

The increase in carboxyl methylation induced by guanosine 5',3-O-(thio)triphosphate (GTPgammaS) in brush border membranes from rat kidney cortex was studied, and the methyltransferase activities affected by this nucleotide analog were identified. Addition of GTPgammaS to brush border membranes stimulated the carboxyl methylation in a time-dependent manner while adenosine and guanine nucleotides were ineffective. The GTPgammaS-dependent carboxyl methylation was inhibited by the chelating agents EDTA (63%) and 1,10-phenanthroline (68%), suggesting that this activity required divalent cations. The methyl ester groups induced by the addition of GTPgammaS to brush border membranes were unstable, with about 80% of them hydrolyzed following 1 h incubation at 37 degrees C. The GTPgammaS stimulation of the carboxyl methylation in brush border membranes was unaffected by the detergent 3-[(3cholamido)-dimethylammonio]-1-propanesulfonic acid up to a concentration of 0.4% (w/v). At this latter detergent concentration, the activity of prenylated protein methyltransferase (PPMT) was strongly inhibited and that of l-isoaspartyl/d-aspartylmethyltransferase (PIMT) was increased twofold, as measured with their respective exogenous substrates, N-acetyl-S-farnesyl cysteine and ovalbumin. GTPgammaS increased the methylation of several substrates in brush border membranes. The induced methylation in substrates migrating between 20 and 36 kDa was strongly decreased by the competitive inhibitor farnesylthioacetic acid, a synthetic farnesylated substrate for PPMT, while a delta-sleep-inducing peptide containing an L-isoaspartyl residue inhibited that of substrates with molecular weights above 36 kDa, suggesting that PIMT activity was also involved. This interpretation was strengthened by the observation that the increased methylation induced by GTPgammaS in these membrane substrates was completely lost following their analysis by gel electrophoresis under alkaline conditions. Taken together, these results indicate that both PPMT and PIMT activities are regulated by guanine nucleotides in brush border membranes of rat kidney.

Topics: Animals; Chelating Agents; Cholic Acids; Enzyme Activation; Enzyme Inhibitors; Esters; Farnesol; Guanosine 5'-O-(3-Thiotriphosphate); Kidney; Male; Microvilli; Nucleotides; Ovalbumin; Protein D-Aspartate-L-Isoaspartate Methyltransferase; Protein Methyltransferases; Protein O-Methyltransferase; Rats; Rats, Sprague-Dawley

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.

Topics: Animals; Cell Membrane; Cholic Acids; Chromatography, Gel; Chromatography, Ion Exchange; Kidney Cortex; Kinetics; Methylation; Microvilli; Molecular Weight; Ovalbumin; Protein D-Aspartate-L-Isoaspartate Methyltransferase; Protein Methyltransferases; Rats; Substrate Specificity