A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis.

We have studied the post-translational processing of p21ras proteins. The primary translation product pro-p21 is cytosolic and is rapidly converted to a cytosolic form (c-p21) of higher mobility on SDS-PAGE. c-p21 is converted in turn to the membrane-bound mature palmitoylated form (m-p21) of slightly higher mobility. These processing steps are accompanied by increases in isoelectric point and in hydrophobicity as judged by Triton X-114 partitioning. Although the increases in electrophoretic mobility and hydrophobicity precede acylation we show that mutation of Cys186, which has been shown to block acylation, also abolishes the pro-p21 to c-p21 conversion. Thus the Cys186 residue is involved in the processing steps prior to acylation. We have identified two processing events which contribute to the pro-p21 conversion. Site-directed mutagenesis to insert tryptophan, which is not present in the wild type, followed by metabolic labelling with [3H]tryptophan has allowed us to map a proteolytic processing event which removes the three C-terminal residues. In addition, both the c-p21 and m-p21 forms are carboxyl-methylated. Approximately one methyl group is incorporated per molecule of p21 at steady state, which can partially account for the increase in isoelectric point. Unlike palmitate, methyl group turnover is not observed.     

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.     

Response of microbial adhesives and biofilm matrix polymers to chemical treatments as determined by interference reflection microscopy and light section microscopy.

The polymers involved in the adhesion of Pseudomonas fluorescens H2S to solid surfaces were investigated to determine whether differences between cell surface adhesives and biofilm matrix polymers could be detected. Two optical techniques, i.e., interference reflection microscopy (IRM) and light section microscopy (LSM), were used to compare the responses of the two types of polymer to treatment with electrolytes, dimethyl sulfoxide (DMSO), and Tween 20. To evaluate initial adhesive polymers, P. fluorescens H2S cells were allowed to attach to glass cover slip surfaces and were immediately examined with IRM, and their response to chemical solutions was tested. With IRM, changes in cell-substratum separation distance between 0 and ca. 100 nm are detectable as changes in relative light intensity of the image; a contraction of the polymer would be detected as a darkening of the image, whereas expansion would appear as image brightening. To evaluate the intercellular polymer matrix in biofilms, 3-day-old biofilms were exposed to similar solutions, and the resultant change in biofilm thickness was measured with LSM, which measures film thicknesses between 10 and 1,000 microns. The initial adhesive and biofilm polymers were similar in that both appeared to contract when treated with electrolytes and to expand when treated with Tween 20. However, with DMSO treatment, the initial adhesive polymer appeared to contract, whereas there was no change in thickness of the biofilm polymer. These results indicate that both polymers bear acidic groups and thus act electrostatically with cations and are able to enter into hydrophobic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.     

Stoichiometry of cellular and viral components in the polyomavirus middle-T antigen-tyrosine kinase complex.

Our results indicate that only one type of tyrosine kinase is present within each middle-T antigen-tyrosine kinase complex, suggesting that middle-T antigen forms separate complexes with different tyrosine kinases. Furthermore, we determined that there is only one molecule of middle-T antigen within any one of these complexes. We interpret this to mean that in any given cell, polyomavirus transformation involves, at least in part, the simultaneous deregulation of a number of separate pathways controlling cellular proliferation. Finally, we also demonstrate that the separate middle-T:pp60c-src and middle-T:pp59c-fyn complexes are each able to interact with the same cellular p81/85-kDa phosphoprotein, a possible component of the phosphatidylinositol kinase.     

G6PD Huntsville: a new glucose-6-phosphate dehydrogenase associated with chronic hemolytic anemia

Summary We describe a previously unreported glucose-6-phosphate dehydrogenase (G6PD) variant. G6PD Huntsville was found in a Caucasian male, resident of Huntsville, Alabama who was investigated for otherwise unexplained chronic hemolytic anemia. An unusual feature of this unique, apparently hemolytic, G6PD mutant is that its red cell enzymatic activity has not been decreased. The mutant enzyme is unstable. Additionally, the enzyme variant is characterized by normal electrophoretic mobility, biphasic and slightly alkaline pH optimum, and abnormal kinetics for the natural substrates G6PD and NADP as well as the artificial substrates deamino NADP. Its activity for another artificial substrate 2-deoxy G6PD is normal. The inhibition constant for NADPH is normal. The subject has had no evidence of episodic jaundice.     

Genome instability in interspecific cell hybrids I. Unstable expression of genes in divergent cell hybrids

Somatic cell hybrids between cells of widely divergent mammalian species display a range of chromosomal and genetic anomalies which may be the equivalent of the “genomic shock” phenomena observed in many plant and animal interspecific hybrids. Mouse-kangaroo hybrids show extreme segregation and fragmentation of the kangaroo chromosomes. Here 1 show that, in addition to the chromosomal instability, some hybrids display unstable expression of three genes borne on the kangaroo active maternal X chromosome. These genes (HPRT, G6PD andPGK) may be co-ordinately inactivated at high frequency, then reactivated once more. I suggest that this reversible inactivation in interspecific hybrids may be the result of an unstable change at an X inactivation centre located in the kangaroo X_q.