Assembly of F1-ATPase in isolated mitochondria

The assembly of the proton-translocating ATPase complex was studied in isolated mitochondria by incubating yeast mitochondria with radiolabeled precursors of mitochondrial proteins which had been made in a cell-free protein synthesis system. Following such an incubation, the ATPase complex (F1F0) was isolated. Newly assembled F1-ATPase was detected by autoradiography of the isolated enzyme, only peptide subunits which had been made in vitro and imported into the isolated mitochondria could be radioactive. Incorporation of radiolabeled ATPase subunits into the enzyme does not occur in the presence of an uncoupler of oxidative phosphorylation or of a divalent metal chelator, nor does it occur in submitochondrial particles rather than intact mitochondria. Incorporation of labeled ATPase subunits into the enzyme can be completed by unlabeled subunits, provided the unlabeled proteins are added before the mitochondria are incubated with radioactive precursors. These findings suggest that F1-ATPase is assembled from a pool of subunits in mitochondria.     

Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin

This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.