Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.     

The destruction of spores of Bacillus subtilis by the combined effects of hydrogen peroxide and ultraviolet light

Ultraviolet light irradiation of bacterial spores in the presence of hydrogen peroxide has been shown to produce synergistic kills when compared with ultraviolet light (u.v.) and hydrogen peroxide used sequentially. This use in combination has been patented for the commercial sterilization of packaging before filling with UHT-processed products. Previous results have shown that lamps producing u.v. light with a maximum output at about 254 nm were extremely effective. Results obtained using a Synchrotron radiation source to produce a narrow band of irradiation now shows that the greatest kill of spores of Bacillus subtilis in the presence of hydrogen peroxide is obtained with radiation at ˜270 nm. Such results suggest that the action of the u.v. light is not directly on the spore DNA but may be related to the production of free hydroxyl radicals from hydrogen peroxide.     

Involvement of protease in L-glutamine control of nucleic acid and polyphosphate metabolism in cells transformed by 9,10-dimethyl-1,2-benzanthracene, SV40 and H-ras oncogene

Mammalian cells transformed with either 9,10-dimethyl-1,2-benzanthracene, SV40 or H-ras oncogene dramatically changed their ability to synthesize DNA and RNA and metabolize polyphosphate when L-glutamine was withdrawn from the growth medium or when heat shocked (growth at 42 degrees C). Untransformed, DNA and RNA synthesis decreased by 50-80% when glutamine was withdrawn, but polyphosphate accumulated whether or not glutamine was supplied. Heat shock did not alter this response. Transformed isogenic cells responded differently; at 37 degrees C, they decreased their synthesis of DNA and RNA if starved for glutamine, whereas at 42 degrees C, synthesis was optimal without glutamine. Transformed cells accumulated polyphosphate at 37 degrees C when starved for glutamine, but at 42 degrees C, no polyphosphate accumulated. This apparent non-dependence on glutamine by transformed cells when heat shocked was found to be due to the production of glutamine from serum proteins through induction of a protease(s).     

An approach to chemotaxonomy of the Asarum subgenus

A survey of chemical composition of 23 species of Asarum subgenus Heterotropa showed that the five groups could be distinguished on the basis of the presence or absence of asatone, phenol ethers and terpenes.     

The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12.

We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA. We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R. Freudl, H. Schwarz, M. Klose, N. R. Movva, and U. Henning, EMBO J. 4:3593-3598, 1985). A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed. In this construct the signal sequence was fused to the periplasmic part of the protein. The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm. Hence, information required for export does not exist within the OmpA protein.     

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)