Ectoprotein kinase activity of the isolated rat adipocyte.

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ-³²P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10^?5 M and 7.14 pmoles/min/1.5 × 10⁵ cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.     

Genetics of formamidase-5 (brain formamidase) in the mouse: Localization of the structural gene on chromosome 14

A single formamidase, which is different from the formamidases found in other tissues, occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity: For-5 ^b is relatively heat stable and is present in strain C57BL/6J, while For-5 ^d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studied to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5 ^d allele is associated with the Es-10 ^b allele, and that the For-5 ^b allele is associated with Es-10 ^a and Es-10 ^c. Formamidase-5 segregates as expected in the F₂ generation of crosses between strains bearing For-5 ^b and For-5 ^d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.This research was sponsored in part by the Department of Energy under contract with the Union Carbide Corporation and in part by NIH Research Grant GM-18684 from the National Institute of General Medical Sciences. J. C. F. is a predoctoral Fellow supported by Grant CA 09104 from the National Cancer Institute. The Biology Division of Oak Ridge National Laboratory and the Jackson Laboratory are fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Role of β-carotene in the reaction centres of Photosystems I and II of spinach chloroplasts prepared in non-polar solvents

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl₄, n-heptane) and the β-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b₆ in the ratio 1:2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP⁺ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn²⁺ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of β-carotene in photochemistry separate from its roles in energy transfer and photoprotection.Removal of the fraction of β-carotene closely associated with the Photo-system I reaction centre caused the rate of NADP⁺ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of β-carotene, photochemistry can still be observed, however the specific association of β-carotene with the reaction centre is required for maximal rates. We propose that β-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state β-carotene and chlorophyll in the first excited state.     

Neurite formation and membrane changes of mouse neuroblastoma cells induced by valinomycin

A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K⁺ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells.     

Early hCG-induced desensitization in Leydig cells.

The early mechanism of hCG induced down regulation of its own receptor as well as steroidogenesis refractoriness of rat Leydig cells to gonadotropin stimulation have been investigated. A single injection of 5, 12, 25, 50 and 100 IU of hCG in rats induced within 8 hours, Leydig cells desensitization. However, apparent receptor loss was significantly lower only in the rats who received 50 and 100 IU of hCG. Cycloheximide inhibits hCG-induced receptors loss but had no effect on hCG-induced desensitization. The most likely explanation for desensitization in the presence of binding sites and a normal adenylate cyclase, is a defective coupling between the receptor sites and the catalytic subunit.     

Antimetabolic activities of 2-fluoro-L-histidine.

2-Fluoro-L-Histidine inhibits protein synthesis in various cell cultures, as measured by ³H-leucine incorporation. This histidine analog also inhibits the cytopathogenicity of a number of RNA and DNA viruses in primary and continuous cell cultures; it blocks the transformation of normal mouse (MO) cells by murine sarcoma virus, and partially suppresses the release of murine leukemia virus by a continuously infected mouse cell line (JLSV5). In human skin fibroblasts, it reduces the interferon-inducing capacity of poly(I)·poly(C). Inhibition of cell protein synthesis may be the common cause of the various effects. 4-Fluoro-L-histidine is essentially inert in all of the test systems examined.