α-Helix-to-random-coil transitions of two-chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross-linked selectively at C-190

A method is described for preparation of a species of β tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion-exchange chromatography. (4) The C36-blocked β chains are refolded by dialysis. (5) The refolded, C36-blocked ββ species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, C190-cross-linked ββ product is separated from contaminating species—mostly completely blocked β-chains and multichain cross-linked molecules—by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO₄/polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked ββ species and for its DTT-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190-cross-linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the physics.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

The regulation of exogenous NAD(P)H oxidation in spinach (Spinacia oleracea) leaf mitochondria by pH and cations.

Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation of exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low at pH 7.0, even in the presence of 1 mM-CaCl2. There was a proportional increase in the apparent Km for NADPH with decreasing H+ concentrations, suggesting that NADPH protonated on the 2'-phosphate group was the true substrate. Exogenous NADH was oxidized by oxygen with an optimum at pH 6.9. Under low-cation conditions, EGTA or EDTA (both 1 mM) had no effect on the Vmax. of NADH oxidation, although the removal of bivalent cations from the membrane surface by the chelators could be observed by use of 9-aminoacridine fluorescence. In contrast, under high-cation conditions, chelators lowered the Vmax. by about 50%, probably due to a better approach of the negatively charged chelators to the negative membrane surface than under low-cation conditions. In a low-cation medium, the Vmax. of NADH oxidation was increased by about 50% by the addition of cations. This was caused by a lowering of the size of the negative surface potential through charge screening. In contrast with other cations, La3+ inhibited NADH oxidation, possibly through binding to lipids essential for NADH oxidation. The apparent Km for NADH varied 6-fold in response to changes in the size of the surface potential, suggesting that the approach of the negatively charged NADH to the active site is hampered by the negative surface potential. The results demonstrate that the spinach leaf cell can regulate the mitochondrial NAD(P)H oxidation through several mechanisms: the pH; the cation concentration in general; and the concentration of Ca2+ in particular. The results also emphasize the importance of electrostatic considerations when investigating the kinetic behaviour of membrane-bound enzymes.     

Effect of the beta-gamma phosphate bond of ATP on synthesis of leader RNA and mRNAs of vesicular stomatitis virus.

RNA products synthesized in vitro by vesicular stomatitis virus with normal nucleotides or imido analogs were compared by polyacrylamide gel electrophoresis. When imido ATP, which has a nonhydrolyzable beta-gamma bond, was substituted for ATP, leader RNA and DI particle product were synthesized, but appreciable mRNA synthesis was not detected.