Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum

Carbon monoxide dehydrogenase (CO dehydrogenase) from Rhodospirillum rubrum was shown to be an oxygen-sensitive, nickel, iron-sulfur, and zinc-containing protein that was induced by carbon monoxide (CO). The enzyme was purified 212-fold by heat treatment, ion-exchange, and hydroxylapatite chromatography and preparative gel electrophoresis. The purified protein, active as a monomer of Mr = 61,800, existed in two forms that were comprised of identical polypeptides and differed in metal content. Form 1 comprised 90% of the final activity, had a specific activity of 1,079 mumol CO oxidized per min-1 mg-1, and contained 7 iron, 6 sulfur, 0.6 nickel, and 0.4 zinc/monomer. Form 2 had a lower specific activity (694 mumol CO min-1 mg-1) and contained 9 iron, 8 sulfur, 1.4 nickel, and 0.8 zinc/monomer. Reduction of either form by CO or dithionite resulted in identical, rhombic ESR spectra with g-values of 2.042, 1.939, and 1.888. Form 2 exhibited a 2-fold higher integrated spin concentration, supporting the conclusion that it contained an additional reducible metal center(s). Cells grown in the presence of 63NiCl2 incorporated 63Ni into CO dehydrogenase. Although nickel was clearly present in the protein, it was not ESR-active under any conditions tested. R. rubrum CO dehydrogenase was antigenically distinct from the CO dehydrogenases from Methanosarcina barkeri and Clostridium thermoaceticum.     

Effects of viral transformation on synthesis and secretion of collagen and fibronectin-like molecules by embryonic chick chondrocytes in culture

Monolayer cultures of chondrocytes isolated from 11-day-old chick embryo vertebral cartilage were transformed by Rous sarcoma virus, and the effects of transformation on synthesis and secretion of extracellular proteins by these cells were studied. Transformation resulted in decreased synthesis of type II collagen which did not appear to be due to underhydroxylation of collagenous protein but to a decrease in the total amount synthesized. Carboxymethyl-cellulose chromatography and polyacrylamide disc gel electrophoresis failed to demonstrate any alpha 2 chains as a result of the transformation, suggesting that conversion of type II to type I collagen did not occur. In contrast to the decrease in collagen synthesis, synthesis of a molecule with biochemical characteristics similar to fibronectin increased markedly in virally transformed cultures. Although there were no significant differences in the amount of fibronectin-like molecules in the cell layers of normal and transformed chondrocytes, a marked increase of these molecules in the culture media of the transformed cells was demonstrated. These findings were confirmed by experiments with temperature-sensitive mutants of the virus.     

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.     

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.     

Comparison of the effects of smooth and skeletal tropomyosin on skeletal actomyosin subfragment 1 ATPase

Chicken gizzard tropomyosin, like rabbit skeletal tropomyosin, inhibits and activates skeletal actomyosin subfragment 1 ATPase at low and high [subfragment 1], respectively, showing that both smooth and skeletal tropomyosin qualitatively produce similar cooperative effects on activity. For gizzard tropomyosin, however, the extent of the inhibition was less, and the activation curve rose more sharply at lower [subfragment 1]. In terms of a two-state cooperative activity model for the actin-tropomyosin filament (Hill, T. L., Eisenberg, E., and Chalovich, J. (1981) Biophys. J. 35, 99-112), these results qualitatively suggest that, for the gizzard tropomyosin system, more units are initially in the active state (in the absence of subfragment 1) and that the switching of units to the active state is more cooperative. The greater cooperativity indicated for the gizzard system may be a consequence of the greater rigidity of gizzard tropomyosin indicated from conformational studies.     

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.     

The effect of water activity and pH on the production of mycotoxins by fungi growing on a bread analogue

Mycotoxin production by various toxigenic fungi, growing on a bread analogue, was investigated at various water activities (a_w) and pH combinations. Citrinin, ochratoxin A and sterigmatocystin could be detected at a_w > 0·80, while patulin was only observed at a_w= 0·95. These results show that some toxins may be produced at lower water activities than have been reported on synthetic media and suggest that, where possible, natural substrates should be used to investigate factors affect-ing mycotoxin production in foodstuffs.