Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.     

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol P_i/min per 10⁸ ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the ‘carrier structure’ for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.     

Species-specific aggregation factor in sponges. IV. Inactivation of the aggregation factor by mucoid cells from another species.

The role of protein synthesis in the cell union was investigated in conjugation of Blepharisma intermedium. In order to avoid possible complications due to the occurrence of other processes in conjugation, the homotypic cell union, in which conjugation is arrested at the stage of cell union without further changes, was used. Such unions were induced by treating cells of one mating type with the gamone of the other mating type for about 2 h. The induction of cell union was regularly accompanied by increased protein synthesis, which started 5 min after the beginning of the gamone treatment and continued for about 2 h. When protein synthesis was inhibited by cycloheximide, cell union was also inhibited. The extent of the two inhibitions were closely correlated. We concluded that gamone induces proteins and that protein synthesis is essential for cell union. Proteins synthesized in gamone-treated and non-treated cells were also separated and compared. Consideration of these results leads to a hypothesis that most of the gamone-induced proteins are membrane proteins normally synthesized, though in lesser amount, in non-conjugating cells and that cells gain the capacity to unite when these proteins are accumulated at a restricted area on the cell surface by another gamone-controlled mechanism.     

Structure-function relationships of shortened [LeuB25]insulins, semisynthetic analogues of a mutant human insulin

Replacement of B25-phenylalanine by leucine in the insulin sequence causes marked inactivation. The effect of this sequence variation was studied here in des-(B26-30)-insulin. [LeuB25]des-(B26-30)-insulin and its B25-amide were prepared by trypsin-mediated semisynthesis from N-terminally protected des-(B23-30)-insulin and synthetic tripeptides. The relative lipogenic potency in isolated rat adipocytes was 8.0% for the truncated analogue with a free B25-carboxyl function, and 18.1% for the amidated analogue. Binding to cultured human IM-9 lymphocytes was 4% and 9%, respectively. Thus, both shortened insulins are markedly more active than [LeuB25]insulin. The PheB25----LeuB25 substitution in both the shortened and the full sequence has a moderate effect on the CD spectrum, indicating that the gross main chain conformation is largely retained in both molecules. Independent of the substitution an absolute increase of the circular dichroism is observed upon amidation of the B25-carboxyl group.     

The synthesis and some biological properties of N-(6-purinyl)peptides

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. III. The determination of 14C-labeled 6-thiopurines in L5178Y cell extracts using high-pressure liquid cation-exchange chromatography.

A method is presented for the separation of 6-thiopurine bases and ribonucleosides, of sulphate anions and of common purine bases and oxidized purines by means of high-pressure liquid cation-exchange chromatography using a 0.18 X 100 cm column, filled with Beckman M71 resin, and eluted with 0.4M ammonium formate, pH 4.6, at a linear flow velocity of 5.2 cm/min at 50 degrees C. The method has been applied to the separation and quantitative determination of 14C-labeled 6-mercaptopurine metabolites in HClO4 extracts of L5178Y murine lymphoma cells. Distribution patterns of 14C radioactivity within the cells after a 24 h incubation period with (8-14C)-labeled 6-mercaptopurine have been established. The indentification of 6-mercaptopurine metabolites, such as 6-thioxanthosine ribonucleotide, 6-thioinosinic acid, 6-thioguanylic acid, 6-methylthioinosinic acid, and 6-thiouric acid, after the digestion of the extracts with alkaline phosphatase has been confirmed using the behaviour of each compound in enzymatic peak-shifting analyses with purine nucleoside phosphorylase and the corresponding elution volumes of 6-thiopurine bases and ribonucleosides as proofs. According to the specific radioactivity of the (8-14C)-labeled 6-mercaptopurine batch, the amounts of the various 6-mercaptopurine metabolites in about 6% of the total HClO4 extract of 1.6 . 10(8) labeled cells have quantitatively been determined as 1--130 pmol. The intracellular concentration of 6-thiopurines was determined at 1.4 . 10(-5)mol/1.