Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of the hexose-monophosphate pentose pathway by delta 1-pyrroline-5-carboxylic acid in human fibroblasts.

L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.     

Single copy DNA homology in sea stars

Summary The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA fromPisaster ochraceus was reannealed with excess genomic DNA fromP. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, andDermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines.P. brevispinus DNA contains essentially all of the sequences present inP. ochraceus single copy tracer whileEvasterias andPycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeledEvasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology betweenP. ochraceus single copy DNA andSolaster orDermasterias DNA. SimilarlySolaster DNA contains sequences homologous to approximately 18% ofDermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the generaPisaster andEvasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The twoPisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the generaPisaster andEvasterias.     

Directly observed 15N NMR spectra of uniformly enriched proteins

The proteins cytochrome c2, cytochrome c', and ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum were enriched in 15N by growth of the organism on 15NH4Cl. The proteins were purified to homogeneity and studied by 15N NMR. Longitudinal and transverse relaxation times as well as the nuclear Overhauser effects were determined for various groups of the proteins which vary in molecular weight from 13,000 to 114,000. The values of these parameters for the amide resonances or for groups thought to be rigid were consistent with the molecular weights of the proteins. Relaxation times of the amino-terminal alpha-amino groups and the side chain nitrogen atoms of arginine and lysine were consistent with much more rapid motion. Nitrogen atoms having bound protons were generally found to be decoupled from the protons by chemical exchange. Demonstrable 1H-15N coupling was taken as an indication that exchange was hindered, either by hydrogen bonding interactions or by inaccessibility of the group to solvent. Histidine side chain nitrogen atoms, which experience a large chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical exchange and tautomerization. Strategies for improving sensitivity and for obtaining specific peak assignments are also discussed.     

A small one-band paracentric inversion inv (4) (p15.3p16.3).

A neonate with aniridia was found to have a one band paracentric inversion of the short arm of chromosome 4. This was initially difficult to interpret on high resolution banding. The inversion was present in three generations of the family.     

Characterization of p51/52, a cell-growth regulated protein of WI-38 cells.

A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52-kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53.     

Cardiopathic effects of dichloroacetate in the fetal Long-Evans rat.

Dichloroacetic acid (DCA) is a by-product of the chlorine disinfection of water and may occur in treated water at levels exceeding 100 micrograms/L. Previous studies revealed teratogenic effects, particularly heart malformations, at high doses (900-2,400 mg/kg given on days 6-15 of pregnancy). In a series of three studies, groups of 7-10 Long-Evans rats were dosed with 1,900 mg/kg of DCA on days 6-8, 9-11, or 12-15; with 2,400 mg/kg on days 10, 11, 12, or 13; and with 3,500 mg/kg on days 9, 10, 11, 12, or 13, in an attempt to determine the most sensitive period and further characterize the heart defect. In a fourth study, six dams were treated with 1,900 mg/kg of DCA days 6-15 of pregnancy, and 56 fetuses were harvested for light microscopy of the heart. Eight control fetuses from four litters were also examined. No heart malformations were seen in the groups treated with 1,900 mg/kg DCA days 6-8 but were present in the group treated on days 9-11 and 12-15, with the higher incidence occurring on days 12-15. Single doses of 2,400 mg/kg DCA given on days 10, 11, 12, or 13 resulted in a much lower incidence of cardiac malformations, which occurred only on days 10 and 12. The high dose of DCA (3,500 mg/kg) did not increase the incidence of heart defects but showed that dosing on day 9 as well as on days 10 and 12 would produce the defect. The defects seen were characterized as high interventricular septal defects (H-IVSD). Light microscopy showed that the defect was caudal to the semilunar valves, with the anterior right wall of the aorta communicating with the right ventricle. Another aspect of the defect is at the level of the semilunar valves, with the right cusp or sinus of Valsalva in communication with the right ventricle. The defects are discussed more fully and methods for further study suggested.