The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Modulation of the transferrin receptor during DMSO-induced differentiation in HL-60 cells

When HL-60 cells are induced to differentiate by dimethyl sulfoxide along a granulocytic pathway there is a fivefold decrease in the total number of transferrin receptors within 3 days, as compared to untreated cells. This decrease is due primarily to a rapid decline in the synthesis of the receptor rather than an increase in the degradation of the receptor. The decrease in transferrin receptor synthesis is a specific and early event that precedes the cessation of cell proliferation, differentiation, and the decrease in total protein synthesis.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease.

Four genes (ssp genes) coding for small, acid-soluble spore proteins of Bacillus megaterium and the gene for the protease that cleaves them during germination were cloned in the integratable plasmid pJH101. Each plasmid was integrated into the B. megaterium chromosome by a Campbell-type mechanism, allowing mapping of all five genes. The gene for the small, acid-soluble spore protein-specific protease (gpr) mapped near rib, and the sspA gene mapped between argA and hisA. The three other genes of the spp gene family (sspB, -D, and -F) all mapped near metC/D, with the order: sspF-sspD-metC/D-hemA-argO-sspB. While neither gpr nor sspF has been mapped in B. subtilis, the positions of the sspA, -B, and -D loci are similar in B. megaterium and B. subtilis, suggesting that the members of this multigene family have not recently undergone significant movement on the chromosome. It appears that more gene rearrangement has occurred in the flanking genes than has occurred in the ssp family of genes producing the small, acid-soluble spore proteins.     

A disulfide-bonded short chain collagen synthesized by degenerative and calcifying zones of bovine growth plate cartilage

Studies of collagen synthesis by specific sections of individual fetal bovine costochondral junction growth plates were conducted and histologically related to the zones from which the sections were derived. Sections were metabolically labeled in organ culture to examine the synthesis of collagen and its precursors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Type II collagen was the major species synthesized in all tissue sections; 1 alpha, 2 alpha, 3 alpha collagen chains were synthesized in all growth plate sections and to a small extent in the fetal structural cartilage. A short chain collagen was synthesized predominantly in the zones of degeneration and provisional calcification and accounted for 8-12% of the radioactivity in this section. This short chain collagen has 63-kDa subunits which are converted to 46-kDa species by limited proteolysis with pepsin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that both the pepsin- and non-pepsin-treated forms of short chain collagen are disulfide-bonded. Digestion with bacterial collagenase showed that the 46-kDa and a major portion of the 63-kDa forms are collagenous. Pulse-chase studies in organ culture did not demonstrate an obvious precursor to the 63-kDa form, and there was no conversion to the 46-kDa after 20 h. Synthesis of short chain collagen appears to be specific to the process of endochondral ossification in the growth plate; its appearance may be critical to this transition process.     

Coh A,a mutation affecting the post aggregative related (par) cohesive system of Dictyostelium discoideum

Three stage-specific cohesive systems operate in D. discoideum: VEG, elaborated by vegetative cells: AR, by aggregation competent cells; and PAR, by post aggregation stage cells. Previous study of a mutant strain JC-5 had shown the stability of its PAR system (but not the AR) to be temperature sensitive. However, the phenotypic expression of this mutation termed Coh A is complicated by the presence in that strain of a preexisting mutant gene Rde A, which accelerates developmental events generally and alters the pattern of morphogenesis. Genetic evidence presented here indicates that the two mutations have been separated by parasexual recombination yielding a Coh A, Rde A⁺ segregant class of which strain JC-36 is a prototype. At the permissive temperature, JC-36 follows a morphogenetic sequence like that of the wild type in respect to timing, morphogenetic pattern, and spore appearance. At the restrictive temperature, it forms normal aggregates at the usual time but exhibits two morphogenetic aberrancies during post aggregative development. First, fruit construction is arrested at a stage approximating the 16 hr “Bottle” stage of the wild type, though more squat and blunt tipped, and then the aggregate regresses. Cytodifferentiation into spores and stalk cells is also blocked. Second, a shift of slugs migrating normally at the permissive temperature to the restrictive causes the latter to disintegrate progressively as they leave clumps of cells behind them within the flattened sheath. JC-36 cells developing at the restrictive temperature also exhibited a decrease in EDTA resistant cohesivity attributable on two grounds to the sensitivity of the PAR system. In addition, the disappearance of the AR system completed in the wild type by the Mexicanhat (18–19 hr) stage is indefinitely arrested at an intermediate level in JC-36.     

Molecular basis for the special properties of proteins and enzymes from Halobacterium marismortui

Abstract Three proteins from Halobacterium marismortui , malate dehydrogenase (hMDH), glutamate dehydrogenase (hGDH) and ferredoxin (hFD) were purified and characterized with respect to their molecular masses, amino acid composition and, for hFD only, primary structure. Striking features of halophilic proteins are: the high excess of acidic over basic residues; acidic clusters in the sequence. Low-salt concentration causes inactivation and changes in structural parameters of hMDH and hGDH. Reactivation of hMDH involves long-lived stable intermediates. The salt concentration optimum of enzymic activity is independent of salt nature. The high capacity of halophilic proteins to retain water and salt is due to unique molecular properties, studied by physico-chemical techniques.