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The uptake of tritium-labeled [D-Ala2,D-Leu5]enkephalin ([3H]DADLE) by mouse neuroblastoma cells (N4TG1) was investigated under conditions which are optimal for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was a receptor-mediated process, since it was inhibited by opiate receptor ligands and the (i) time course, (ii) dose-response curve, and (iii) temperature dependence of uptake were similar to those for enkephalin-receptor down regulation. Cells in suspension showed less uptake than those in monolayer culture and both uptake and down regulation were decreased by the inhibitors of metabolic energy production, sodium azide, and 2,4-dinitrophenol. Comparison of the effects of these metabolic inhibitors on the processes of receptor loss and ligand uptake showed that these cells accumulate [3H]DADLE in excess of their surface receptor number, suggesting that receptor recycling normally occurs under the conditions studied. The lysosomotrophic amines, chloroquine and methylamine, inhibited dissociation of cell-associated [3H]DADLE but did not affect down regulation. The data are consistent with the idea that enkephalin is internalized via receptor-mediated endocytosis. The possible fate of the "down-regulated" receptors is considered.

Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 10--9 M to 10--10 M) of a highly radioactive (125-I, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 125-I-Labeled insulin, cholera toxin and the plant lictins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Plasma membranes are labeled before homogenization by incubating intact cells briefly at 24 degrees or 4 degrees, or by very brief in situ perfusion of the organ, with the 125-I-Labeled marker. After removing the free 125-I-labeled ligand from the medium by washing (at 4 degrees), the membrane-marker complex remains intact over prolonged (days) periods of time at 4 degrees. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. When desired, the complex can be dissociated deliberately by manipulating experimental conditions such as temperature or by adding specific simple sugars. The most generally suitable marker appears to be WGA. At least in certain tissues (e. g. fat cells) labeling of the plasma membrane with 125-I-WGA and 125-I-isnulin can be performed equally well and selectively in homogenates as in the intact cell. 125-I-Cholera toxin cannot be used in homogenates because of significant binding to nuclei. The use of 125-I-labeled WGA as a specific plasma membrane marker is illustrated in following the course of fractionations, and in quantitating the yield and purity, of plasma membranes from fat cells, lymphocytes, and liver. The results are compared with simultaneous measurements of the plasma membrane enzyme "markers," ATPase, 5-nucleotidase, and basal as well as hormone-stimulated adenylate cyclase activities. The fractionation of liver plasma membranes by aqueous dextran-polyethylene glycol two-phase polymer systems and by conventional differential centrifugation procedures arealso quantitated with the marker, 125I-WGA. Substantial quantities of plasma membrane material are no recovered in the interphase of the two-phase polymer system. Conventional liver fractionation procedures which retain, for further purification, only the readily sedimented pellet (2000 times g, 15 min) discard a very large (at least 70%) questenal hy

Addition of low concentrations of ATP, but not other nucleoside triphosphates, to fat cells inhibits insulin-stimulated glucose oxidation under conditions that measure glucose transport. Basal rates of glucose transport are not inhibited. The inhibition cannot be overcome by increasing the concentration of insulin, and the dose-response relationships indicate no gross alteration in the apparent affinity of the cells for the hormone. The binding of 125I-labeled insulin is not altered by ATP. Under the conditions used, ATP does not alter oxidation by affecting cytoplasmic metabolic processes. The effects of exogenous ATP on glucose transport were confirmed by direct measurements of 3H-labeled 3-O-methyl-d-glucose uptake in isolated fat cells. ATP inhibits the insulin-stimulated but not the basal rate of 3-O-methylglucose transport. The inhibition by ATP is relatively selective since it represents a fall in the apparent affinity for glucose of the insulin-activated component of the glucose transport system. The inhibition can be overcome by high glucose concentrations. The antilipolytic activity of insulin in ATP-treated cells is unaffected while insulin-stimulated transport is markedly depressed; these activities of insulin can thus be dissociated. ATP also inhibits the enhanced rates of glucose transport observed with concanavalin A, wheat germ agglutinin, spermine, spermidine, cysteine, and glutathione, substances that activate the same glucose transport system as insulin. The ouabain-stimulated glucose oxidation, which occurs by mechanisms different from those of insulin, is not affected by ATP. The ATP analog, α,β-methylene-adenosine 5'-triphosphate (Ap(CH2)pp), can mimic the effect of ATP. Another analog, β,γ-methylene-adenosine 5'-triphosphate (App(CH2)p), does not suppress insulin-stimulated transport but it can effectively inhibit the ATP effect; this phosphonic acid analog cannot donate its terminal phosphoryl moiety in phosphorylation reactions. Phloretin, a strong competitive inhibitor of d-glucose transport, can protect the cells from the ATP effect. Phlorizin, another competitive inhibitor, potentiates the effect of ATP. Fat cells exposed to low concentrations (5 to 50 µm) of ATP for a short time (5 min) at 24° continue to exhibit a diminished insulin response (glucose transport) after thorough washing to remove the ATP in the medium. The recovery of insulin sensitivity during subsequent incubation at 37° is slow, pointing to the protracted and relatively irreversible effect of such mild treatment with ATP. Brief treatment of cells with [γ-32P]ATP results in the selective phosphorylation of two plasma membrane components which are estimated to be of molecular weight 16,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. Phosphorylation of the latter is inhibited by phloretin, and phosphorylation of both is inhibited by App(CH2)p. The possibility is entertained that the procedures utilized may selectively label membrane components involved in insulin-mediated glucose transport (glucose carriers). Consideration is given to the possible physiological significance of such selective membrane phosphorylation in the control of insulin sensitivity in normal as well as insulin-resistant states.

A highly sensitive and specific radioimmunoassay for three enkephalins (opiate-like pentapeptides) has been developed. The assay utilizes /sup 125/I-labeled enkephalins and antisera raised in rabbits, to synthetic enkephalins coupled with glutaraldehyde to bovine serum albumin. These antisera show

The phosphorylation of specific membrane proteins by an endogenous protein kinase has been studied in purified membrane fractions from rat adipocytes using trichloroacetic acid precipitation and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The endogenous phosphorylation of two specific membrane proteins is completely dependent on the presence of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and magnesium ions. This phosphorylation occurs very rapidly at 24°, reaching maximal levels at 1 min. The number of sites specifically phosphorylated in the presence of cyclic AMP probably does not exceed 50,000 per fat cell, requiring the use of very high specific activity (10 to 50 Ci per mmole) [γ-32P]ATP for these studies. The minimal molecular weights of the two specifically phosphorylated proteins are about 22,000 and 16,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The same two proteins are phosphorylated when intact fat cells are exposed briefly to low concentrations of exogenous ATP, a process which results in the suppression of the insulin-stimulated rates of d-glucose transport. At least 95 % of the cyclic AMP-dependent 32P incorporated into trichloroacetic acid-precipitable protein is in the form of protein-bound phosphoserine. The concentration of cyclic AMP required for maximal stimulation of phosphorylation is about 1.5 µm. Cyclic guanosine 3':5'-monophosphate has no significant effect unless its concentration is increased to 10-4 m. Phosphorylation is inhibited by calcium ions. The cyclic AMP-stimulated phosphorylation is inhibited by phloretin and by 5'-adenylyl-β, γ-methylene triphosphonate. The specific cyclic AMP-dependent membrane phosphorylation is also found in membranes from cells of obese rats, an insulin-resistant animal. This phosphorylation system, however, cannot be detected in membranes from guinea pig fat cells. The lack of phosphorylation of specific membrane proteins in guinea pig fat cell membranes is correlated with the insulin insensitivity of this tissue to glucose transport (but not lipolysis) and with the inability of ATP to inhibit the slight stimulation which insulin exerts on glucose transport. The possibility is considered that this specific cyclic AMP-dependent phosphorylating system is involved in the hormonal regulation of glucose transport and the modulation of insulin sensitivity.

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.

The interaction of beta-endorphin with opiate receptors was studied by using the radioiodinated, metabolically stable D-Ala2 derivative of human beta-endorphin. This analog binds specifically to rat brain membrane preparations with an apparent Kd of about 2.5 x 10-9 M. The ability of various enkephalin analogs, as well as opiate agonists and antagonists, to inhibit the binding of beta-endorphin clearly demonstrates that this peptide can bind to opiate receptors. However, the effects of various cations on the binding of 125I-[D-Ala2]beta-endorphin are markedly different from those found for enkephalin binding. Sodium ion at physiological concentrations decreases substantially the binding of enkephalins but only slightly decreases endorphin binding, whereas manganese enhances enkephalin binding but has no effect on endorphin binding. Moreover, potassium (100 mM) decreases the binding of beta-endorphin but does not affect enkephalin binding. These results suggest that beta-endorphin and enkephalin bind differently to the same receptor or bind to different receptors with overlapping specificity.