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P F Pilch

Publications and source records attributed to P F Pilch.

103 records · Page 6Linked to original sources
PubMed1981

Identification of the glucagon receptor in rat liver membranes by photoaffinity crosslinking.

The photoaffinity crosslinker hydroxysuccinimidyl-p-azidobenzoate was used to attach (125)I-labeled glucagon covalently to a rat liver membrane protein of M(r) approximately 53,000. Membranes that had been incubated with (125)I-labeled glucagon were treated in the dark with hydroxysuccinimidyl-p-azidobenzoate, and a covalent complex was then formed by irradiation with ultraviolet light. Characteristics of (125)I-labeled glucagon binding and covalent attachment to the M(r) 53,000 peptide were consistent with this peptide being a component of the glucagon receptor involved in the activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. Binding and covalent attachment of (125)I-labeled glucagon to the M(r) 53,000 peptide were inhibited by glucagon concentrations that were within the dose-response curve for adenylate cyclase activation, and GTP specifically decreased the photoaffinity crosslinking of (125)I-labeled glucagon to the M(r) 53,000 peptides. Insulin did not compete for the photoaffinity crosslinking of (125)I-labeled glucagon. The same technique of photoaffinity crosslinking that covalently attached (125)I-labeled glucagon to the M(r) 53,000 peptide with an efficiency of 1-2% can be used to attach (125)I-labeled insulin covalently to a M(r) 125,000 peptide with an efficiency of approximately 10%. This peptide has been shown to be a subunit of the high-affinity insulin-binding site in rat liver membranes. The technique of photoaffinity crosslinking with agents like hydroxysuccinimidyl-p-azidobenzoate provides a rapid, simple method of covalently attaching ligands to their putative receptors. Photoaffinity crosslinking does not require chemical modification of the labeled ligand and has a less stringent requirement for specific reactive groups than the commonly used bifunctional crosslinking reagents.

Adenylyl Cyclases↗
PubMed1980

Hormone binding alters the conformation of the insulin receptor.

Fat cells or fat cell membranes were briefly subjected to mild proteolysis under conditions where insulin receptors were either free or bound to (125)I-labeled insulin. When receptors were then affinity-labeled to visualize the effects of this treatment, it was observed that receptors that had been occupied by ligand during proteolysis exhibited greater rates of degradation than unoccupied receptors. These results demonstrate that insulin-receptor interaction induces a change in receptor structure that may be related to signal transmission.

Adipose Tissue↗
PubMed1980

The subunit structure of the high affinity insulin receptor. Evidence for a disulfide-linked receptor complex in fat cell and liver plasma membranes.

125I-Insulin equilibrated with high affinity fat cell and liver plasma membrane receptors was cross-linked to the membrane by addition of disuccinimidyl suberate. Autoradiographic analysis of the 125I-insulin-linked membranes subsequent to dodecyl sulfate electrophoresis in the absence of reductant revealed the presence of one labeled species which migrated with an apparent molecular weight of 300,000. Electrophoresis of these membranes in the presence of dithiothreitol resulted in the appearance of one major labeled band of about Mr = 125,000 concomitant with the loss of label in the Mr = 300,000 region. Another radioactive species which migrated in the Mr = 225,000 region on the reduced gels contained a much smaller amount of label. The degree to which the Mr = 125,000 membrane component was cross-linked to 125I-insulin in these experiments paralleled the extent of occupancy of high affinity membrane receptors by the 125I-insulin. 125I-Insulin-linked plasma membranes derived from adipocytes alkylated with N-ethylmaleimide prior to homogenization to prevent spontaneous sulfhydryl oxidation also exhibited the Mr = 300,000 and 125,000 labeled bands upon electrophoresis in the absence and presence of reductant, respectively. These same radioactive species were observed when 125I-insulin was covalently corss-linked to intact adipocytes. These data indicate that the labeled monomer with an apparent molecular weight of 125,00 represents a high affinity insulin receptor subunit which exists in the native adipocyte plasma membrane in a disulfide-linked complex. The amount of cross-linked label present in the reduced form of the receptor (Mr = 125,000) was only 20 to 30% of that present in the oxidized species (Mr = 300,000). Since the 125I-insulin used in these studies is labeled predominantly on the A chain, this large loss of label upon reduction indicates that it is predominantly the B chain of insulin that is covalently linked to the receptor protein by disuccinimidyl suberate. 125I-Proinsulin which lacks an A chain NH2 terminus could also be cross-linked to the receptor, indicating that the A1 glycine is not critical to the cross-linking reaction. 125I-Insulins modified either at lysine B29 with an acetyl group or at phenylalanine B1 with a phenylthiocarbamoyl group were both readily cross-linked to the receptor with roughly equal efficiency. It is concluded that the B1 terminal amino and B29 lysine amino groups are both accessible for cross-linking of insulin to the receptor by disuccinimidyl suberate.

Adipose Tissue↗
PubMed1980

Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.

Plasma membrane insulin receptors, affinity labeled by covalent crosslinking to receptor-bound 125I-labeled insulin, are shown to appear as a heterogeneous population of three major disulfide-linked complexes (Mr 350,000, 320,000, and 290,000) upon electrophoresis in highly porous dodecyl sulfate/polyacrylamide gels in the absence of reductant. This pattern is consistent in all rat and human tissues that were analyzed. Upon reduction of disulfide bonds, each of these receptor structures is dissociated in two successive steps. Low concentrations of dithiothreitol promote a first step of disulfide bond reduction in which the Mr 350,000 species splits into a Mr 210,000 form and the Mr 290,000 species splits into a Mr 160,000 form. In contrast, both the Mr 210,000 and Mr 160,000 receptor fragments are generated from the native Mr 320,000 species upon partial reduction, indicating an asymmetrical structure. The second step of receptor reduction occurs upon treatment of the native disulfide-linked receptor complexes with high concentrations of dithiothreitol. Under these conditions, the Mr 350,000 receptor yields a Mr 125,000 subunit, denoted as alpha, and a Mr 90,000 subunit, denoted as beta, whereas the Mr 290,000 receptor dissociates into the alpha subunit and a Mr 49,000 subunit, denoted as beta 1. The Mr 320,000 receptor band is found to consist of alpha, beta, and beta 1 subunits upon complete reduction. The partially reduced Mr 210,000 receptor fragment is composed of the alpha subunit disulfide-linked to the beta subunit, whereas the Mr 160,000 species consists of the alpha subunit disulfide-linked to the beta 1 subunit. Thus, the stoichiometry of the three ubiquitous native insulin receptor structures of Mr 350,000, 320,000, and 290,000 are (alpha) 2 (beta) 2, (alpha) 2 (beta) (beta 1), and (alpha) 2 (beta 1) 2, respectively.

Animals↗
PubMed1980

Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulin-treated adipocytes.

The cis-monoenoic fatty acids vaccenate and oleate stimulate D-glucose transport when partitioned into isolated plasma membranes from rat adipocytes. The magnitude of hexose transport stimulation due to these agents is equal to that observed in plasma membranes derived from insulin-treated adipocytes. Addition of cis-unsaturated fatty acids to plasma membranes derived from insulin-treated cells results in no further stimulation of glucose transport over that due to the hormone alone. In contrast, treatment of membranes exhibiting insulin-activated D-glucose transport activity with saturated fatty acids reduces transport activity to control levels. No effect of the saturated fatty acids was observed on D-glucose transport in control membranes. Because cis-unsaturated fatty acids fluidize plasma membranes under the conditions used in these experiments, these data demonstrate a positive correlation between membrane fluidity and adipocyte D-glucose transport system activity. In addition, the results suggest that enhanced bilayer fluidity or increased affinity of the glucose transporter for fluid microenvironments of the membrane may play a key role in transport regulation by insulin.

Adipose Tissue↗
PubMed1980

Effect of thyroid status on insulin action in rat adipocytes and skeletal muscle.

Isolated adipocytes and soleus muscles prepared from mature rats, rendered hypothyroid by a low iodine diet and propylthiouracil, markedly resisted the ability of insulin to increase glucose utilization. In adipocytes, the sum of basal d-(1-(14)C)-glucose conversion to CO(2), glyceride-glycerol, and fatty acid was unaltered by hypothyroidism, although conversion to fatty acid was decreased. The response of each of these metabolic pathways to insulin at all concentrations tested was greatly diminished in hypothyroid rat adipocytes. 3-O-Methylglucose transport rates in the presence of insulin were not significantly different in adipocytes from hypothyroid as compared with euthyroid rats, although basal transport rates were significantly higher in the hypothyroid state. Lipolysis and cyclic AMP accumulation in adipocytes from hypothyroid rats in response to theophylline were markedly diminished compared with euthyroid controls, but insulin was about as effective in inhibiting lipolysis in these cells as in those derived from euthyroid animals. The binding of (125)I-insulin to adipocytes at several hormone concentrations was also shown to be unaffected by hypothyroidism. In soleus muscle, basal glucose conversion to H(2)O and glycogen was unaltered in the hypothyroid state, whereas insulin action on these pathways was markedly inhibited. The decrease in muscle insulin responsiveness was less marked than that observed in adipocytes. Uptake of either 2-deoxyglucose or l-arabinose in the presence or absence of insulin was similar in soleus muscles derived from euthryoid vs. hypothyroid rats. Similarly, insulin action on the conversion of soleus muscle glycogen synthase D to the I form in the absence of glucose was unaltered by hypothyroidism. We conclude that (a) hypothyroidism in mature rats leads to a marked decrease in the responsiveness of glucose metabolism in adipocytes and skeletal muscle to insulin; (b) no detectable impairment of the membrane insulin effector systems that mediate the regulation of adipocyte hexose transport and glycogen synthase is caused by hypothyroidism in this animal model; and (c) the cellular defect that leads to apparent insulin resistance of adipocyte and soleus muscle glucose utilization resides at the level of one or more intracellular enzymes involved in glucose catabolism.

Adipose Tissue↗
PubMed1976

Fluorine-containing analogues of intermediates in the Shikimate pathway.

The phosphoenolpyruvate analogue (Z)-phosphoenol-3-fluoropyruvate is a substrate for phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase from Escherichia coli. In the presence of excess erythrose 4-phosphate, apparent KM values of 65 and 38 muM were observed for phosphoenol-3-fluoropyruvate and phosphoenolpyruvate, respectively. Because the apparent Vmax for phosphoenol-3-fluoropyruvate is only 1.17% of that for phosphoenolpyruvate, one can study the former as an inhibitor of 3-deoxy-arabino-heptulosonic acid-7-phosphate synthase. Kinetic experiments showed phosphoenol-3-fluoropyruvate to be competitive with respect to phosphoenolpyruvate. Two distinguishable Ki values of 8 and 48 muM were obtained. The product (3S)-3-deoxy--3-fluoro-arabino-heptulosonic acid 7-phosphate was purified, characterized, and shown to act as a substrate for 5-dehydroquinate synthase. 3-Deoxy-3-fluoro-arabino-heptulosonic acid 7-phosphate, in contrast to 3-deoxy-arabino-heptulosonic acid 7-phosphate reacts slowly or not at all with reagents specific for 2-keto-3-deoxy sugars and is relatively resistant to oxidative cleavage by sodium periodate. The expected product of periodate oxidation, 3-fluoro-3-formylpyruvate, cannot be detected. This observation was clarified by studies with model compounds.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMedDate not supplied

Stimulation of tyrosine-specific phosphorylation in vitro by insulin-like growth factor I.

Several mitogens elicit tyrosine-specific protein kinase activities. Although the physiological significance of this is unclear, the generality of these reactions implies that this may be an inherent feature of growth factor-growth factor receptor interactions. The observed mitogenic properties of the polypeptide insulin-like growth factor I (IGF-I) indicated that it might also stimulate such activity. We report here that IGF-I stimulates a tyrosine-specific protein kinase in a time- and dose-dependent fashion. The close correspondence between an approximate 50% effective dose (ED50) of phosphorylation and an approximate Kd for IGF-I binding leads us to conclude that a high-affinity IGF-I receptor, not the structurally similar insulin receptor, is the mediator of IGF-I stimulated kinase activity. Immunoprecipitation indicates that both the beta-subunit of the IGF-I receptor and the beta-subunit of the insulin receptor are targets for the IGF-I-stimulated protein kinase.

Enzyme Activation↗