Differential sensitivity to guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions

The effects of GTP gamma S on glucose transport activity reconstituted from adipocyte membrane fractions were studied in order to test the hypothesis that intrinsic activity changes of the insulin-sensitive glucose transporter may be mediated by guanine nucleotide-dependent mechanisms. GTP gamma S and GTP inhibited reconstituted glucose transport activity by 50% in membrane fractions from insulin-treated cells in a concentration-dependent manner; no inhibitory effect was observed in membrane fractions obtained from basal cells. GDP, GMP and guanosine were less effective than GTP, whereas the adenine nucleotides ATP gamma S and AMP failed to reduce the reconstituted transport activity. The data indicate that guanine nucleotides may modulate the activity of the adipocyte glucose transporter. Since the effect is dependent on treatment of cells with insulin, the hormone appears to induce a specific functional alteration of the glucose transporter

Bidirektionale hormonale Modulation spannungsabhängiger Ca2+-Kanle [Bidirectional hormonal modulation of voltage dependent ca2+ channels]

The role of guanine nucleotide-binding proteins (G-proteins), acting as transducers between membranous receptors activated by extracellular signals and enzymatic effectors controlling the concentrations of intracellular signal molecules, is well established. G-proteins are also involved in the hormonal modulation of voltage-dependent Ca2+ channels. In various cell types, the increase in intracellular signal molecules via G-protein-coupled receptors causes activation of protein kinases which may stimulate or inhibit voltage-dependent Ca2+ channels. For example, voltage-dependent Ca2+ channels of cardiac and skeletal myocytes are stimulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase. Other protein kinases, i.e., cyclic guanosine monophosphate (cGMP)-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase C, also appear to be involved in the hormonal modulation of Ca2+ channels. According to this principle, G-proteins exert a distant control of ion channel activity. In addition, there appears to exist another mechanism which does not involve intracellular signal molecules or protein kinases stimulated by intracellular signal molecules. The only signal transduction components identified so far include receptors, G-protein and Ca2+ channels. Ca2+ channel modulations following this apparently membrane-confined mechanism have been described to occur in neuronal, endocrine and cardiac cells. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis-toxin-sensitive G-protein, possibly G0. The G-protein involved in the hormonal stimulation of Ca2+ channels in adrenocortical and pituitary cells may represent a pertussis-toxin-sensitive G-protein of the Gi-type. The choleratoxin-sensitive G-protein, Gs, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step

Phosphorylation of G-protein alpha-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects

The phosphorylation of G-protein alpha-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [32P]phosphate and subsequently incubated in the presence or absence of insulin. In iodinated or unlabeled plasma membranes, antiserum generated against a peptide corresponding to a region common to G-protein alpha-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as Gs and Gi alpha-subunit, respectively, by comparison with [32P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [32P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [32P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with G alpha s, since (1) the peptide used to raise the antiserum failed to inhibit significantly immunoprecipitation of the 45 kDa phosphoprotein with the antiserum, (2) in contrast to the Gs alpha-subunit, the phosphoprotein was readily removed from the immunocomplex by washing with sodium dodecyl sulfate (SDS), and (3) the subcellular localization of the phosphoprotein differed considerably from that of the Gs alpha-subunit. No phosphate was detected in immunoprecipitates from either basal or insulin-treated cells after the 45 kDa phosphoprotein had been removed. These data argue against a mediating role of phosphorylated G-protein alpha-subunits in the action of insulin

Immunochemical detection of the alpha-subunit of the G-protein, GZ, in membranes and cytosols of mammalian cells

An antiserum (AS 98) was raised against a synthetic peptide deduced from published cDNA sequences of the alpha-subunit of the putative G-protein, GZ (Fong et al. Proc. Natl. Acad. Sci. USA 85, 3066-3070, 1988; Matsuoka et al. Proc. Natl. Acad. Sci. USA 85, 5384-5388, 1988). In membrane and cytosol preparations of many but not all tested mammalian tissues, AS 98 predominantly recognized two proteins of 40 and 43 kDa Mr. Whereas high levels of a 40 kDa GZ alpha-subunit were found in rat liver membranes and in brain cytosol, AS 98 failed to detect the alpha-subunit of GZ in brain membranes

Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels

Analyse dynamischer Vorgaenge an technischen Oberflaechen mit Speckle-Korrelation

Changes in the micro-profile of rough surfaces are analysed by correlation of digital images of speckle patterns that arise from laser light scattering at the surface. The quantitative interpretation of decorrelations is based on a theoretical model and its verification by alternative measurements on sample objects. Profile changes of some 10 nm are well resolved. The scope of applications is illustrated by a corrosion process on an iron probe and the removal of a patina layer from a stone by laser ablation. (orig.)Available from TIB Hannover: DtF QN1(76,48) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go

Antisera generated against synthetic peptides were used to identify G-protein alpha-subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs alpha-subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi alpha-subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2 alpha-subunit. A serum specific for the Go alpha-subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go