Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

Changes in the size and shape of the synaptic vesicles in the sensorimotor cortex of the rat brain in the initial phases of kindling

The sensorimotor area of rat cerebral cortex was subjected to repeated electrical stimulation at 10-min intervals, with resultant formation and progressive lengthening of self-sustained after-discharges (SSAD). One and 60 min after the third SSAD ended, we carried out an electron microscopy morphometric analysis of the agranular synaptic vesicles in type I synapses (after Gray) in the second cortical layer of the homotopic area of the unstimulated hemisphere. One minute after the seizure ended, 5.8% enlargement of the synaptic vesicles compared with the control was demonstrated in zone II of the synapse (0.1-0.2 micron from the active zone of the synapse). Neither the size nor the shape of the synaptic vesicles in the other parts of the synaptic apparatus altered. Sixty min after the seizure ended, a 5.5% enlargement of the synaptic vesicles in zone I (0.0-0.1 micron) and a 5.4% enlargement of those in zone II was found. The synaptic vesicles in zone I in the experimental animals were more oval than in the controls. Our findings support the vesicular theory and testify that hyperfunction, up to temporary exhaustion of the synaptic apparatuses, produces a change in the transmitter content of the synaptic vesicles. A raised amount of transmitter in the synaptic vesicles near the active zone could be one of the factors responsible for continued hyperexcitability of the tissue one hour after the seizure had ended. The results likewise support the concept of two mechanisms of synaptic vesicle formation, and hence of the existence of two different vesicle populations.     

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.     

Neutrophil-stimulating activity of staphylococci based on reactive chemiluminescence data

The neutrophil-stimulating properties of 38 S. aureus strains and 32 S. epidermidis strains were studied in the reaction of luminol-mediated chemiluminescence. All S. aureus strains and 29 S. epidermidis strains were found to possess neutrophil-stimulating activity, the mean activity index for S. aureus being significantly higher. The stimulating activity of the strains varied within a wide range (the variation coefficient was 120.0 +/- 21.9%) and did not correlate with the content of protein A in bacterial cells and the degree of their hydrophoby. The opsonization of staphylococci with normal human serum enhanced the neutrophil reaction 1.5- to 100-fold and simultaneously leveled out the chemiluminescence indices in experiments with different strains (the variation coefficient was 8.0 +/- 1.5%). The nature of the neutrophil-stimulating effect of staphylococci and its relationship to the exploratory reactions of phagocytes are discussed.     

Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS, whose product is similar to a sigma factor

In many species of actinomycetes, carotenogenesis can be photoinduced. The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency. In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration. We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S. setonii ISP5395 cells the capacity to synthesize carotenoids. Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis. We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S. setonii ISP5395.