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In membranes of S49 lymphoma eye- variants, which lack a functional guanine nucleotide-binding component (Ns protein) mediating adenylate cyclase stimulation by hormones, guanine nucleotides decreased the forskolin-stimulated adenylate cyclase activity by maximally 40 - 60%. The potency order of the guanine nucleotides studied was guanosine 5'-[γ-thio]triphosphate(GTP[S]) ≫ guanosine 5'-[β,γ-imido]triphosphate > GTP > guanosine 5'-[β- thio]diphosphate (GDP[S]). GTP and GDP[S] acted as partial inhibitors; they competitively antagonized the GTP[S]-induced inhibition, which was half-maximal and maximal at about 3nM and 100 nM GTP[S], respectively. Cholera toxin did not enhance the inhibitory action of GTP. The eye- adenylate cyclase inhibition by GTP[S] occurred after a short lag period and persisted after washing the membranes. The inhibition was not affected by the pH (6.5 - 9.0) of the incubation medium and was largely independent of the concentrations of MgATP (up to 200 μM) and Mg2+ (up to 10 mM). In contrast, Mn2+ potently reduced the GTP[S]-induced eye- adenylate cyclase inhibition. Similarly as observed with forskolin, GTP[S] decreased the eye- adenylate cyclase activity stimulated by purified. preactivated Ns protein. Apart from Mn2+, inhibition of the eye- adenylate cyclase by GTP[S] was prevented or reversed by various treatments, which have been shown to obliterate hormone-induced adenylate cyclase inhibition in other cell types, such as by N-ethylmaleimide, by limited proteolysis with trypsin and by a factor extracted from bovine sperm particles. The data indicate that in membranes of eye- variants, which lack a functional N, protein, a guanine nucleotide-binding component (N1) is present, which mediates adenylate cyclase inhibition by guanine nucleotides. Such a component apparently also mediates adenylate cyclase inhibition by hormones in other cell types. Comparison of adenylate cyclase stimulation and inhibition by guanine nucleotides suggests that N1 is activated and inactivated by similar but not identical mechanisms as the Ns protein and that the relative activity states of these coupling components determine the activity of the adenylate cyclase. [ABSTRACT FROM AUTHOR]

In cultured lines of mouse lymphoma cells, resistance to glucocorticoids is frequently associated with the occurrence of glucocorticoid receptors with an abnormally low affinity (nt-) or an abnormally high affinity (nti) for nuclei and DNA. We have investigated whether the abnormal affinities for DNA are correlated with alterations m charge and surface properties of the receptors, that would be revealed through the partition coefficient in aqueous dextran/poly(ethylene glycol) two-phase systems. We have found that none of the receptor variants is defective in the activation step per se, and that only the nti receptors are abnormal in partition properties. Partial proteolysis of wild-type and nt- receptors with α-chymotrypsin produces forms which are indistinguishable from the nt- receptors with respect to partition coefficients. Upon a-chymotrypsin treatment the wild-type receptors attain DNA-binding properties identical to those of the nti receptors, while the nt- receptors, in spite of some increase in DNA affinity, still bind less firmly to DNA than the α-chymotrypsin-treated wild-type receptors. α-Chymotrypsin treatment of the various receptor types also produces an increase in the binding to dextran sulphate, but the dextran sulphate affinity is higher and varies less between different receptor types than the DNA affinity. Trypsin-treated receptors were found to be devoid of affinity for DNA and dextran sulphate. We conclude that the increased DNA affinity of the nti receptors is due to a gross change in the structure of the receptors, while the decreased DNA affinity of the nt- receptors is caused by more subtle alterations in the polyanion binding domain of the receptors. [ABSTRACT FROM AUTHOR]

Thiolase from pig heart has been carboxymethylated in 6 M guanidine-HCl, and the N-terminal sequence of the carboxymethylated protein is shown to be uniquely Val-Ser-Lys, A trypsin digest Ires been examined by peptide mapping techniques and the number of peptides (42-46) detected is in good agreement with the total number of lysine and arginine residues calculated from the amino acid composition of the enzyme tetramer and assuming 4 identical chains per 170000 g of protein. No evidence for the presence of more than one type of subunit was found when both the untreated and the carboxymethylated proteines were examined by polyacrylamide and starch gel electrophoresis in 6 M urea. From these results it is proposed that thiolase is composed of four very similar and probably identical protein chains each comprising approximately 400 amino acids including one catalytically active cysteine. [ABSTRACT FROM AUTHOR]

Inactivation of thiolase with iodo[1-14C] acetamide has been shown to be due to its reaction with cysteine residues in the enzyme. [1-14c] Carbamolmethyleysteine was identified as the only product of the reaction and a study of the radioactive peptides that were isolated from a tryptic digest of the [1-14C]carbamoylmethyl-enzyme has shown that the reactive cysteines occur in a unique sequence extending to at least 26 amino acid residues in the primary structure. The enzyme-substrate compound formed when thiolase reacts with [1-14C]acetyl-CoA is shown to contain [14C]acetyl groups bound in thioester linkage to at least three cysteines per mole. Moreover these residues of [14C]acetyl-cysteine occur exclusively in a sequence that is identical to the sequence around the [14C]carbamoymethylcysteine residues in the enzyme-inhibitor compound, showing that the substrate and the inhibitor react with the same four cysteine residues in the enzyme. These results provide additional evidence for the chemical identity of the four subunits of thiolase. [ABSTRACT FROM AUTHOR]