PubMed Health⌕ Search

Biomedical subjects

M D Hollenberg

Publications and source records attributed to M D Hollenberg.

At least 145 records · Page 8Linked to original sources
PubMed1985

Distinct hydrodynamic forms of the insulin receptor: electrophoretic analysis of the RI and RII species.

In previous work, we identified two insulin receptor species, RI (KAV = 0.31) and RII (KAV = 0.53), that could be separated by gel filtration on Sepharose 6B. In the present study, we sought to establish that these two receptor species do represent larger (RI) and smaller (RII) oligomeric forms of the receptor, rather than representing receptor species separated from each other by differential adsorption to the Sepharose matrix. Receptor solubilized from isolated human placenta membranes was purified by lectin- and insulin-agarose chromatography and was radiolabeled with carrier-free 125I. The labeled receptor was separated by Sepharose 6B gel filtration into two fractions (peak I, KAV = 0.31; peak II, KAV = 0.53), was immunoprecipitated by anti-insulin receptor antibody, and was analysed by electrophoresis in nonreducing polyacrylamide slab gels. The autoradiograms of the gels indicated that peak I (KAV = 0.31, RI receptor form) contained a number of receptor species of 240 000 daltons or greater, whereas peak II (KAV = 0.53, RII receptor form) contained mainly receptor species of 210 000 daltons or smaller. In particular, large amounts of a 90 000 dalton species (presumably free receptor beta-subunit) were present in peak II. Incubation of the material obtained from peak I with insulin resulted in a change in the electrophoretic pattern, which became identical with that observed for material recovered from peak II.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1985

Vascular actions of epidermal growth factor-urogastrone: possible relationship to prostaglandin production.

Epidermal growth factor-urogastrone (EGF-URO), a polypeptide of about 6000 atomic mass units present in humans, mice, rats, and other mammals that is widely recognized for its mitogenic and acid-inhibitory activities, was observed to cause contraction of helical arterial strips of the rat ileocolic artery and to potentiate the action of KCl on helical strips from the superior (cephalic) mesenteric artery. The contractile effect of EGF-URO in ileocolic preparations displayed marked tachyphylaxis at high EGF-URO concentrations (100 ng/mL). The apparent mean effective concentration for EGF-URO in these tissues was about 10 ng/mL (1.7 nM); the detection of EGF-URO degradation in the organ bath indicated that a half-maximal response may have been achieved at a concentration as low as 50% of this value (i.e., about 0.9 nM). The actions of EGF-URO were abolished by indomethacin (3 microM) and were, in part, mimicked by prostaglandin F2 alpha (PGF2 alpha), which contracted the ileocolic preparation and potentiated the action of KCl in the superior mesenteric preparation. In the superior mesenteric preparation, PGF2 alpha alone, like EGF-URO, did not have an appreciable effect on resting tension. The time lag of the EGF-URO effect in the ileocolic preparation (5-6 min to reach peak contraction) corresponded closely with the time course of EGF-URO-stimulated calcium accumulation measured previously in other cell systems. Our results suggest that EGF-URO acts to modulate contractility in vascular tissue via a cyclooxygenase pathway product (possibly PGF2 alpha). Our data point to a role for EGF-URO and possibly other growth factors in the control of vascular function.

Alprostadil↗
PubMed1985

Action of insulin modulated by pertussis toxin in rat adipocytes.

We studied the effect of pertussis toxin (PT) treatment on the ability of insulin to inhibit lipolysis and to stimulate glucose oxidation in isolated rat adipocytes. In cells maximally modified by PT (100% ADP ribosylation of a 41-kdalton protein in membranes), the ability of insulin to inhibit lipolysis stimulated either by PT alone or in combination with a catecholamine was abolished. In cells wherein ADP ribosylation was submaximal (about 67% modification), a small but variable antilipolytic action of insulin could still be detected. In cells maximally modified by PT, both basal and insulin-stimulated glucose oxidation were markedly reduced (to 10-15% of control levels). However, relative to the basal oxidation level, the fold stimulation by insulin in PT-treated cells was equivalent to the fold stimulation in control cells. Nonetheless, PT treatment caused a rightward shift in the dose-response curve for insulin-stimulated glucose oxidation as well as a small reduction in insulin binding. Our results point strongly not only to a link between the inhibitory guanine nucleotide regulatory complex (Gi) and the antilipolytic action of insulin but also to a link between the Gi complex and the overall regulation of glucose metabolism in adipocytes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.

Amino Acids↗
PubMed1984

Connective tissue activation. XXVIII. A connective tissue activating peptide from human urine.

A protein factor in human urine which has the ability to activate connective tissue cells has been identified and partially purified; it appears to be different from epidermal growth factor and IgG. This urinary connective tissue activating factor (CTAP-U) is nondialyzable, labile to protease, stable to thiols, heat, and acid, and has an acidic isoelectric point. Purified preparations of CTAP-U have biologic activities that cause human connective tissue cells to synthesize incremental amounts of 14C-hyaluronic acid, 35S-proteoglycans, and 3H-DNA in vitro. The cell spectrum responsive to this substance includes human synovial cells, human chondrocytes, and skin fibroblasts. CTAP-U does not react with antisera to connective tissue activating peptide-III or to antibodies against IgG or its Fc and Fab fragments. Furthermore, CTAP-U does not cross-react in a radioreceptor assay for insulin, basic somatomedin, or epidermal growth factor-urogastrone. Utilizing standardized isolation conditions, CTAP-U preparations with these properties have been isolated from the urine of 6 normal individuals.

Cartilage↗
PubMed1984

Somatomedin-like activity from cultured embryo-derived cells: partial characterization and stimulation of production by epidermal growth factor (urogastrone).

We have measured the appearance in cell culture media of a somatomedin-like activity (SLA) that cross-reacts in a basic-somatomedin radioreceptor assay and radioimmunoassay and that is produced by cultures of normal fetal-derived human (WI-38 fibroblasts) and mouse (embryonic palate) tissues and by cultures of a Simian virus 40 transformed WI-38 fibroblast cell line (WI-38 VA13/2RA subline). In addition to human growth hormone (hGH), epidermal growth factor (urogastrone) (EGF-URO), human placental lactogen (hPL), and porcine insulin (INS) were able to stimulate the production of SLA in serum-free cultures of the normal WI-38 fibroblasts; human prolactin (hPRL) caused only a marginal stimulation of SLA production in these cultures. In order of the magnitude of the stimulation of SLA production, the effects of the several hormones tested were EGF-URO greater than INS congruent to hPL greater than hGH much greater than hPRL. The half-maximal stimulatory effect of EGF-URO in the WI-38 cultures was observed at a concentration of about 1 nM. In the mouse palate organ cultures, EGF-URO also stimulated SLA production; the effect of EGF-URO was more pronounced in palate organ cultures derived from day 13 embryos compared with tissue obtained from embryos at days 14 and 15. The SLA produced by the human VA13/2RA subline was characterized further by gel filtration under acid conditions, by Procion dye column chromatography, and by high pressure liquid chromatography (HPLC).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Production of somatomedin-like activity by human adult tumor-derived, transformed, and normal cell cultures and by cultured rat hepatocytes: effects of culture conditions and of epidermal growth factor (urogastrone).

We have measured the production of a basic-somatomedin-like activity (SLA) by a variety of human tumor-derived, transformed, and normal postnatal cell cultures; and we have compared the production of SLA by these cell types with the production of SLA by adult rat hepatocytes cultured in serum-free medium. Cells derived from a human epidermoid carcinoma (KB), a pancreatic carcinoma (Panc-1), a Simian virus 40 transformed adult human skin-derived cell line (SV40 fibroblasts), and a normal adult human skin-derived fibroblast line released SLA when cultured in a serum-free growth medium. No SLA was recovered from the culture medium of human choriocarcinoma-derived cells (BeWo) or of a human lymphoblastoid cell line (IM-9). The production of SLA by rat hepatocytes cultured in serum-free medium appeared to exceed the production of SLA by the other cell cultures. In cultures of KB cells, SV40 fibroblasts, and rat hepatocytes, the production of SLA depended on the frequency with which the growth medium was renewed; in general, the highest rates of SLA production were observed when the medium was renewed every 48-72 h. The presence of mouse epidermal growth factor (urogastrone) (EGF-URO) in the serum-free culture medium stimulated the production of SLA by KB cells and by rat hepatocytes, but did not increase SLA production by normal or by SV40-transformed human skin-derived fibroblasts. We conclude that tumor-derived cells are capable of producing somatomedin-like activity and that the production of SLA by such cells can be subject to controls (nutrient availability, EGF-URO stimulation) that regulate SLA production, either by normal adult tissues, like liver, or by a variety of normal embryonic tissues.

Animals↗
PubMed1983

Epidermal growth factor-urogastrone action: induction of 2',5'-oligoadenylate synthetase activity and enhancement of the mitogenic effect by anti-interferon antibody.

Epidermal growth factor-urogastrone (EGF-URO), early in the course of stimulating DNA synthesis in quiescent human fibroblasts, also causes a three to five-fold elevation of the activity of intracellular 2',5'-oligoadenylate synthetase (2,5A synthetase), an enzyme that has been previously implicated in the antiproliferative effects of interferon. The increase in synthetase activity precedes DNA synthesis by approximately six hours, with maximal synthetase activity either preceding or coinciding with maximal DNA synthesis. EGF-URO stimulation does not result in the secretion of detectable amounts of interferon (IFN) into the growth medium and anti-human IFN-beta antibodies do not block the EGF-URO-mediated rise in 2,5A synthetase activity. Thus, the elevation of 2,5A synthetase can be attributed to the action of EGF-URO itself, and not to IFN. Nonetheless, in the presence of anti-human interferon-antibodies, the time course of EGF-URO-stimulated DNA synthesis is prolonged both in human and in Syrian hamster embryo fibroblasts; the effects of the antibody were reversed in both cell strains by the addition of human IFN-beta (HuIFN-beta). The data suggest a role for 2',5'-oligoadenylate synthetase in the process of EGF-URO-mediated mitogenesis and point to the possible production of interferon-related cell-associated regulators during the course of EGF-URO action.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Receptors for insulin and basic somatomedin: immunological and affinity-chromatographic cross-reactivity.

Human placental receptors for insulin and for human basic somatomedin (BSM, analogous to human insulinlike growth factor I (IGF-I] were selectively cross-link labelled with 125I-labelled insulin and BSM using disuccinimidyl suberate under conditions whereby cross-linking of 125I-labelled insulin to the BSM receptor and of 125I-labelled BSM to the insulin receptor was avoided. We have found that the cross-link-labelled receptors for insulin and BSM, present in equivalent amounts in human placenta membrane preparations, both cross-react with antibodies directed against purified rat liver insulin receptor. Compared with the human insulin receptor, the BSM receptor cross-reacted with the antireceptor antibody to a level of roughly 9%, indicating a limited sequence homology between the insulin and BSM receptors. Since both receptors are present in comparable amounts in solubilized placenta membrane preparations, we sought methods for the selective purification of both receptors from such extracts, so as to provide a basis for further comparative structural studies of the two receptors. We have observed that both receptors were adsorbed by affinity columns of insulin-agarose, in a manner that did not yield insulin receptor entirely free from the BSM receptor. As an alternative for receptor purification, we have found that immunoaffinity columns using antiligand antibody should provide a means for the selective isolation of cross-link labelled receptor from tissues in which both are present in equivalent amounts.

Chromatography, Affinity↗
PubMed1983

Receptors, acceptors, and the action of polypeptide hormones: illustrative studies with epidermal growth factor (urogastrone).

The action of polypeptide hormones is discussed in the context of observations made both with receptors, like the ones for epidermal growth factor (urogastrone) (EGF-URO), insulin, and gonadotropin-releasing hormone (GnRH), and with acceptors, like the ones that participate in the adsorptive pinocytosis of substances like cholesterol (low density lipoprotein acceptor), cobalamin (transcobalamin-II acceptor), and asialoglycoproteins (galactose acceptor). The overall processes that both pharmacologic receptors and acceptors undergo subsequent to ligand binding comprise (a) microclustering; (b) aggregation at both coated ("coated pits") and uncoated plasma membrane regions; (c) internalization, with the formation of endocytic vesicles (endosomes or receptosomes); and (d) either recycling of endosomes (receptosomes) to the plasma membrane or fusion of endosomes (receptosomes) with lysosomes, accompanied by proteolytic degradation of the internalized ligand-receptor complex. The relevance of these processes to hormone action is discussed. Further, illustrative studies with EGF-URO are used to highlight general mechanisms that are thought to participate in the action of a variety of peptide hormones. The microclustering event and the concomitant receptor-regulated membrane phosphorylation reactions are emphasized as key plasma-membrane-localized processes that are very likely involved in the rapid (minutes to tens of minutes) of many peptide hormones. For the delayed actions of hormones (hours to tens of hours; e.g., DNA synthesis), the potential importance of nonlysosomal receptor processing, mediated by SH-requiring proteases that generate intracellular receptor fragments, is pointed out. A scheme for the action of EGF-URO is outlined and a model for peptide hormone action is presented that illustrates how multiple chemical mediators of hormone action may result from the combination of a single hormone with its unique receptor.

Animals↗
PubMed1983

Oxytocin resistance in Brattleboro rat adipocytes and comparative studies on insulin or oxytocin responsiveness in normal rat adipocytes.

We have evaluated factors, other than genetic, which might be related to the lack of an oxytocin-mediated insulinlike response (glucose oxidation; lipogenesis) in adipocytes from Brattleboro rats, homozygous for the diabetes insipidus trait (HoDI rats). The manoeuvres used in an attempt to restore the glucoregulatory responses to oxytocin in HoDI cells (increased glucose in the fat pad digestion medium; increased calcium concentration in the oxidation assay; estrogen treatment; use of [1-14C]glucose as substrate; inclusion of adenosine in the assay medium; vasopressin replacement therapy) uniformly failed to result in oxytocin activation of HoDI adipocytes. In contrast, the contractile responses of estrogenized HoDI rat uteri were indistinguishable from those of estrogenized normal rats. We conclude that the nonresponsiveness of the Brattleboro adipocytes to the glucoregulatory actions of oxytocin is not due to factors related to the conditions of the bioassay. On the other hand, in normal fat cells (from Sprague-Dawley and Long Evans rats), oxytocin responsiveness was augmented by a number of the manoeuvres mentioned above, most notably by the inclusion of either calcium (10 mM) or adenosine (10 microM) in the assay medium. Nonetheless, the maximum oxytocin responsiveness of adipocytes from Long Evans or Sprague-Dawley rats, under all conditions of assay, was still only a fraction (less than 20%) of the maximal response to insulin. The effect of adenosine on oxytocin action (increased sensitivity, without an effect on the maximum response) is in keeping with the previously observed effects of this nucleoside on the action of insulin; our results thus pointed to a new parallel in the action of insulin and oxytocin.

Adenosine↗
PubMed1982

Inability of oxytocin to activate pyruvate dehydrogenase in the Brattleboro rat.

Oxytocin has insulin-like activity in that it stimulates lipogenesis and increases pyruvate dehydrogenase activity. However, in adipocytes from homozygous Brattleboro rats oxytocin is incapable of stimulating lipogenesis or pyruvate dehydrogenase activity, although insulin stimulation of both processes is normal and the antilipolytic activity of oxytocin is normal. Thus, the Brattleboro rat provides a new genetic model for the study of oxytocin action, wherein recognition of the chemical mediator is partially defective.

Adipose Tissue↗
PubMed1982

Comparative studies on human placental insulin and basic somatomedin receptors.

The disuccinimidyl suberate, affinity-labeling procedure, and proteolytic mapping techniques have been employed to characterize further the human placental receptors for insulin and basic somatomedin. Electrophoretic analysis of the basic somatomedin receptor, selectively crosslinked to 125I basic somatomedin in the presence of excess native insulin revealed, under reducing conditions, major labeled constituents of 270-280 and 125-140 kd, substantiating our previous work employing a photoaffinity labeling reagent. Affinity labeling also demonstrated the presence of less intensely labeled components with apparent molecular weights of 40 and 45 kd but failed to reveal a distinct 90- to 100-kd species observed in parallel experiments with insulin. In the absence of beta-mercaptoethanol, all components specifically labeled with 125I basic somatomedin migrated in the 300- to 400-kd range. In comparison, selective affinity labeling of the insulin receptor in the presence of excess native basic somatomedin revealed components, upon electrophoresis under reducing conditions, with apparent molecular weights of 270-280, 125-140, 90-100, and 40 kd. The major insulin-labeled component (125-140 kd) comigrated with the major constituent (125-140 kd) selectively labeled with basic somatomedin. When digestion was performed prior to solubilization, chymotryptic and tryptic proteolysis of the membrane-localized selectively labeled insulin, and basic somatomedin receptors yielded quite similar gel electrophoretic maps. However, when digestion was done subsequent to solubilization, chymotryptic and tryptic proteolysis of selectively labeled insulin and basic somatomedin receptors solubilized in SDS yielded similar but not identical gel electrophoretic maps. We conclude that the receptors for basic somatomedin and insulin are highly homologous structures with respect to their disulfide crosslinked composition, and with respect to the size of the major components detected by selective affinity-labeling procedures. Nevertheless, the detection of electrophoretically distinct labeled receptor components upon analysis of specifically labeled intact or proteolytically digested receptors points to subtle differences between the polypeptide compositions of the two receptors.

Disulfides↗
PubMed1982

Characterization of the human placental receptor for basic somatomedin.

We have determined optimal conditions for the solubilization of the basic somatomedin (SM) receptor from human placental membranes and for the measurement of the binding of basic SM to the solubilized receptor. Further, we have developed conditions under which the basic SM receptor, in the presence of equivalent amounts of insulin receptor, can be selectively and specifically affinity-labeled with 125I-labeled basic SM, using the cross-linking reagent disuccinimidyl suberate (DSS). Our results with these developed methods indicate that the properties of the soluble basic SM receptor (pH optimum for ligand binding, pH 7 to 9; adsorption to lectin-agarose derivatives; sedimentation coefficient in detergent-sucrose solutions, 11S) closely parallel data previously reported for the insulin receptor. Based on the sedimentation coefficient and the previously estimated Stokes radius of the soluble receptor (7.2 nm), a molecular weight of 402 000 can be calculated for the detergent-receptor complex. Electrophoretic analysis of the basic SM receptor, selectively cross-linked to 125I-labeled basic SM with DSS in the presence of excess unlabeled insulin revealed, under reducing conditions, a major labeled constituent of 140 kdaltons, substantiating our previous work employing a photoaffinity labeling reagent. DSS cross-linking also demonstrated the presence of less intensely labeled components with apparent molecular weights of 54 000, 43 000 and 35 000 but failed to reveal a distinct 90- to 100-kdalton species visualized in parallel experiments with insulin. The 53-kdalton species was not detected in similar experiments with insulin. A specifically labeled basic SM receptor component of 300 kdaltons was also observed under reducing conditions; in the absence of beta-mercaptoethanol, all labeled components migrated in the 300-kdalton range. In comparison, selective DSS labeling of the insulin receptor in the presence of excess basic SM revealed components which, upon electrophoresis under reducing conditions, exhibited apparent molecular weights of 300 000, 140 000, 90 000--100 000, 43 000 and 35 000. The major insulin-labeled component (140 000) comigrated with the major constituent (140 000) selectively labeled with basic SM. Chymotryptic digestion of the receptors selectively DSS labeled with either 125I-labeled insulin or 125I-labeled basic SM yielded quite similar, but distinctive, gel electrophoretic maps. We conclude that the receptors for basic SM and insulin are highly homologous structures, particularly with respect to their glycoprotein nature, their hydrodynamic properties, their disulphide cross-linked composition, and with respect to the size of the major constituent detected by selective affinity labeling. Nonetheless, the detection of electrophoretically distinct labeled receptor substituents upon analysis of specifically labeled material, both before and after chymotryptic cleavage, points to subtle differences between the polypeptide compositions of the two receptors.

Female↗