Kinetics of acetylation-deacetylation of angiotensin II. Intramolecular interactions of the tyrosine and histidine side-chains

The possible existence of intramolecular interactions involving the tyrosine and histidine residues in angiotensin II has been investigated by measuring the reactivities of the functional groups in the molecule. Angiotensin II catalyzed the hydrolysis of p-nitrophenylacetate in the pH range 6.6-8.2 at higher rates than were consistent with the reactivities of the free constituent functional groups, and had 2-4% of the activity of chymotrypsin between pH 6.6 and 7.5. Treatment of angiotensin II with acetic anhydride demonstrated that the tyrosine hydroxyl and the imidazole side-chain in angiotensin II acetylated and deacetylated at markedly higher rates than for the free amino acids, indicating increased nucleophilicities and the presence of intrinsic deacetylation mechanisms for these residues in angiotensin II. These findings are consistent with the presence of tyrosine hydroxyl-histidine-carboxylate charge relay system in ANG II in aqueous environments, and suggest that ANG II may act at membrane receptors by a mechanism which is analogous to that operating in serine proteases.     

Action of pilocarpine on the normal frog heart and in pathology

Experiments on frogs were performed to examine the effect of the M-cholinomimetic pilocarpine on the heart. It was discovered that at concentrations of 10(-15)--10(-5) g/ml pilocarpine exerted only an adverse chronotropic effect on the perfused heart. When applied at a concentration of 10(-4) g/ml the drug produced a negative as well as a positive chronotropic effect. The latter occurred spasmodically (without progressive rise in the heart rate) in association with a slow heart rate. In some experiments such effects were preceded by a certain deceleration of the heart. In experiments with positive chronotropic effects, arrhythmias and sinoatrial dissociation were observed sometimes. Experiments with recording of the electrograms of the sinuses and lower parts showed that such effects were caused not by pacemaker acceleration but by the removal of the blockade of conduction, between the pacemaker and the atria. As far as the pacemaker is concerned, pilocarpine exerted only a negative chronotropic effect.     

Antibodies against calcium-regulating hormones in infectious-allergic forms of bronchial asthma

Antibodies to calcitonin, parathyroid hormone and cortisol are detected in acute stage of infection-caused bronchial asthma. The appearance of antibodies is paralleled by marked hypercalcaemia. The antibodies may bind excessive hormones in the blood, preventing further hormonal imbalance. Ten-day treatment with glucocorticoids decreased the amount of antibodies possibly due to normalization of hormonal secretion and restoration of their balance. As a result, calcium blood levels returned to normal.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.     

Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

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.     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Location of the isoleucine arrest point in CHO and 3T3 cells

An isoleucine arrest point in G1 was determined by two methods for CHO and 3T3 cells. In the first method the fraction of cells entering S after isoleucine deprivation was assessed by [³H]thymidine labelling and autoradiography. In the second method cells entering S after isoleucine deprivation were identified by double-label autoradiography using [³H] and [¹⁴C]thymidine. From the fraction of cells entering S, determined by the two methods, the arrest point in G1 (and entry into G0) is located within the last 40 min of G1.