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C C Yip

Publications and source records attributed to C C Yip.

At least 55 records · Page 3Linked to original sources
PubMed1986

High molecular weight forms of the insulin receptor.

The insulin receptor of liver, adipose, and placental plasma membranes was photoaffinity labeled with radioiodinated N epsilon B29-(monoazidobenzoyl)insulin. Three specifically labeled bands of 450, 360, and 260 kilodaltons (kDa) were identified in each tissue by polyacrylamide gel electrophoresis of the membranes solubilized in sodium dodecyl sulfate (SDS). The 360- and 260-kDa bands corresponded to partially reduced forms of the 450-kDa band. The distribution of radioactivity between the three insulin receptor bands was dependent on the tissue, the purity of the receptor preparation, and the conditions of solubilization in SDS. The 360- and 260-kDa bands became more prominent in each tissue with an increasing time of solubilization in SDS. However, with a short solubilization time in SDS, the 450-, 360-, and 260-kDa bands of the receptor were distributed approximately in a ratio of 85:15:0 in all three tissues. Inclusion of sulfhydryl alkylating reagents during solubilization in SDS altered this ratio to about 95:5:0. We conclude that the 450-kDa band represents the predominant form of the photolabeled insulin receptor and that the 260-kDa and probably the 360-kDa form as well were generated during the experimental manipulations preceding identification of the receptor. However, the appearance of the 360- and 260-kDa bands was not due to reductant present in SDS or buffer solutions and could not be accounted for by proteolytic degradation of the receptor. Furthermore, purification of the receptor over 2000-fold did not prevent the appearance of the 360- and 260-kDa bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1986

Class I histocompatibility antigens and insulin receptors: evidence for interactions.

We provide evidence for an interaction between mouse class I major histocompatibility complex antigens and insulin receptors. Antibodies against class I but not class II major histocompatibility complex antigens immunoprecipitate photoaffinity-labeled hepatic insulin receptors. Haplotype specificity is demonstrated by reciprocal precipitation using anti-class I antibodies and three strains of mice. Antibodies against the 45-kDa products of either the H-2K or H-2D locus and rabbit anti-mouse beta 2-microglobulin antibodies were shown to precipitate insulin receptors. We also demonstrate the specific binding of 125I-labeled insulin and 125I-labeled epidermal growth factor, but not 125I-labeled glucagon or 125I-labeled atrial natriuretic factor, to solubilized plasma membranes immunoprecipitated with anti-H-2K antibody. These observations suggest a specific interaction between class I major histocompatibility complex antigens and certain hormone receptors.

Animals↗
PubMed1986

Photoaffinity labeling demonstrates binding between Ia molecules and nominal antigen on antigen-presenting cells.

We have used radioiodinated photoreactive bovine insulin as antigen to examine the molecular nature of immunogenic complexes that form on antigen-presenting cells. The probe was allowed to bind to either insulin-presenting B-hybridoma cells, lipopolysaccharide-stimulated blasts, or bovine insulin-specific helper-T-hybridoma cells in the dark. Samples were then exposed to light to induce crosslinkage, solubilized, and analyzed by gel electrophoresis. Two protein bands at about 36 kDa and 27 kDa were specifically labeled on antigen-presenting cells but not on helper T cells. Treatment of these bands with dithiothreitol or endo-beta-N-acetylglucosaminidase F showed that each is composed of a single glycoprotein. These proteins are immunoprecipitable with haplotype-specific but not control anti-Ia antibodies. This identifies the labeled bands as the alpha and beta subunits of class II major histocompatibility antigens. We conclude that a molecular complex may form between Ia and antigen on antigen-presenting cells and that formation of this complex does not require the presence of a helper-T-cell antigen receptor.

Affinity Labels↗
PubMed1985

Peptide mapping of the insulin-binding site of the 130-kDa subunit of the insulin receptor by means of a novel cleavable radioactive photoprobe.

A radioactive photoaffinity probe for the insulin receptor was prepared by derivatizing insulin at its B29 lysine with a novel crosslinking reagent having a cleavable azo linkage. Insulin receptors purified from human placental membranes were photoaffinity labeled with this probe. The photolabeled receptor was treated with dithionite to cleave the azo linkage, thereby removing the insulin ligand and transferring the radioactivity to the receptor protein. The radioactive labeled subunit was isolated and digested with elastase for peptide mapping and separation by high performance liquid chromatography. Results obtained indicated that it will be feasible to use this new photoaffinity probe to obtain radioactive peptides representing the insulin-binding site(s) on the receptor subunit.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of atrial natriuretic factor receptors of rat kidney cortex plasma membranes.

Synthetic rat atrial natriuretic factor (ANF) was derivatized with the N-hydroxysuccinimide ester of [125I]iodoazidosalicylic acid to yield a radioactive photoaffinity probe. Incubation of purified plasma membranes from rat kidney cortex with this photoaffinity probe resulted in the specific labeling of a 140-kDa glycoprotein. The photoaffinity labeling of this protein was inhibited by ANF but not by reduced and alkylated ANF nor by other unrelated peptides. A 140-kDa band was also specifically labeled in liver plasma membranes but not in adipocyte plasma membranes. These observations suggest strongly that the 140-kDa glycoprotein is the ANF receptor.

Adipose Tissue↗
PubMed1985

The preparation and characterization of mono-iodinated photoreactive analogs of insulin.

Photoreactive derivatives of insulin (B29-(p-azidobenzoyl-insulin) iodinated primarily in either the B26 or A14 tyrosine of insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The binding affinities and photoaffinity labeling characteristics of these derivatives were studied in isolated rat adipocytes. Under nonreducing conditions, three forms of the insulin receptor were labeled equally by the B26-derivative, the A14-derivative, and the mixture of the iodinated derivatives. When dithiothreitol was used to reduce the radiolabeled receptors, the radioactivity associated with the binding subunit was much less than that in the intact receptor and the magnitude of the decrease was proportional to the amount of iodine in the A chain of the photoderivatives. Use of the photoreactive derivative iodinated primarily in the B26 position resulted in greater labeling of insulin receptor subunits since most of the radioactivity (80%) remained associated with the receptor upon reduction.

Adipose Tissue↗
PubMed1985

Photoaffinity labelling of hepatic plasma membranes suggests two classes of hepatic insulin receptor.

Photoaffinity labelling of hepatic insulin receptors revealed specifically-labelled bands of 130, 90 and 40 kDa. Endogenous protease activity in hepatic plasma membranes, as well as contaminating proteases present in preparations of clostridial collagenase, degraded some of the 130-kDa insulin-binding subunit to a 115-kDa form. However, a large proportion of the 130-kDa subunits were resistant to degradation, suggesting the presence of two classes of insulin receptor in hepatic plasma membranes. In one class the 130-kDa subunit was sensitive to proteolysis, while in the other it was not. In contrast, the 130-kDa receptor subunits of adipose tissue were all resistant to such degradation. Scatchard analysis of control and collagenase-treated plasma membranes demonstrated that conversion of the 130-kDa subunit to a 115-kDa form did not affect the insulin-binding characteristics of the receptor. It was also apparent that insulin binds to a single class of high-affinity sites in hepatic plasma membranes.

Affinity Labels↗
PubMed1985

Two distinct insulin-related molecules in the guinea pig: immunological and biochemical characterization of insulin-like immunoactivity from extrapancreatic tissues of the guinea pig.

In this study we extracted guinea pig brain and testis with; the extract was adsorbed to and eluted from cartridges (the Sep-Pak C18 procedure). We found this procedure superior for recovering crystalline insulin added to buffers or tissues, and for recovering endogenous insulin from plasma, but inferior for recovery of insulin from tissues. However, we did find 'rat/pork' type-insulin in guinea pig brain and testis (5-50 pg/g wet weight tissue). Our results with the Sep-Pak C18 procedures were reproduced by four other laboratories (who found 4-60 pg/g wet weight of tissue) and similar findings were also obtained by an independent investigator. Thus, we conclude that extrapancreatic tissues of guinea pigs have a second type of insulin-related material that is more typical of other mammalian insulins, but that the amount recovered is dependent upon the extraction procedure utilized.

Animals↗
PubMed1985

Biosynthesis of glucagon (IRG3500) in canine gastric mucosa.

The canine gastric mucosa has previously been shown to contain considerable amounts of a polypeptide with the immunologic and physicochemical characteristics and biologic activity of glucagon (IRG3500). Using mucosal pieces that remained viable for at least 8 h, we have demonstrated that IRG3500 is synthesized in this extrapancreatic tissue. Gel filtration and electrophoresis of extracts of mucosal pieces incubated with 3H-tryptophan, 3H-leucine, or 35S-methionine revealed small amounts of labeled, newly synthesized gastric IRG3500. No labeling of gastric IRG3500 was observed when the mucosa was incubated with 3H-proline, an amino acid not found in glucagon, in the presence of cycloheximide, or in isolated rat hepatocytes. Small amounts of newly synthesized IRG3500 were specifically immunoprecipitated by C-terminally directed glucagon antiserum gamma globulins. The rate of gastric IRG3500 biosynthesis in vitro was apparently unchanged in mucosal pieces from pancreatectomized dogs and unaffected by increased glucose or glucose lack during incubations. Thus we have provided evidence that a hormone of the endocrine pancreas can be synthesized in extrapancreatic tissues.

Amino Acids↗
PubMed1984

The growth of single crystals of (L-arginine)B0 bovine insulin and their preliminary X-ray crystallographic analysis.

The crystals of (L-Arg)B0 bovine insulin large enough for X-ray structure analysis have been grown by vapour diffusion in a buffer containing citrate, acetone and zinc chloride. The X-ray diffraction of the crystals extends to 3.0 A spacing. The crystals belong to the trigonal system with unit cell dimension of a = b = 81.82 A, c = 35.05 A, alpha = beta = 90 degrees, gamma = 120 degrees, space group R3. There are two insulin molecules in the crystallographic asymmetric unit.

Animals↗
PubMed1983

Structure of the insulin receptor of rat adipocytes. The three interconvertible redox forms.

Isolated intact rat adipocytes were photoaffinity labeled with radioactive and photoreactive N alpha B1-monoazidobenzoyl insulin (B1-MABI) or N epsilon B29-monoazidobenzoyl insulin (B29-MABI). Polyacrylamide gel electrophoresis of the labeled plasma membranes solubilized in sodium dodecyl sulfate revealed the specific labeling of three receptor species of 380 kDa, 300 kDa, and 230 kDa. Reduction of each species individually produced the subunits of 130 kDa, 90 kDa, and 40 kDa. Exposure of the adipocytes or plasma membranes after photolabeling to sulfhydryl alkylating agents such as N-ethylmaleimide or p-chloromercuriphenylsulfonate resulted in the appearance of the receptor quantitatively in the 380-kDa form. The effect of the sulfhydryl reagent was concentration dependent and in the case of p-chloromercuriphenylsulfonate the three receptor species reappeared when high concentrations of the reagent were used. Incubation of the adipocytes with low concentrations of dithiothreitol before photolabeling reduced these receptors to discrete lower-molecular-weight forms. In addition, an 85-kDa subunit was now photolabeled by B1-MABI. This subunit was demonstrated to be different from the 90-kDa subunit normally labeled by B29-MABI. We conclude that on the cell surface of the adipocyte, there is one molecular-weight form of insulin receptor of 380 kDa composed of one 130-kDa, one 90-kDa, one 85-kDa, and two 40-kDa subunits. The 300 kDa and 230 kDa are partially reduced forms of the 380-kDa species. We further postulate that a membrane factor or factors sensitive to sulfhydryl alkylating reagents may be involved in the partial reduction and oxidation of these three redox receptor species. The distribution of these redox receptor species may be related to the cellular or tissue sensitivity to insulin.

4-Chloromercuribenzenesulfonate↗
PubMed1983

Insulin receptor: its subunit structure as determined by photoaffinity labeling.

This communication reviews briefly the preparation of two photoreactive arylazido insulins and the use of these insulin derivatives to photolabel the insulin receptor of rat adipocytes. These experiments showed that the receptor contains disulfide linked subunits of 130,000, 90,000, and 40,000 daltons. When not reduced by dithiothreitol, three receptor species of 380,000, 300,000, and 230,000 daltons were detected. Based on the results of photolabeling we propose that the 300,000-dalton species is composed of one 130,000-, one 90,000-, and two 40,000-dalton subunits in disulfide linkages.

Adipose Tissue↗
PubMed1982

Subunit structure of insulin receptor of rat adipocytes as demonstrated by photoaffinity labeling.

Isolated rat adipocytes were incubated in the dark with either one or two radioiodinated photoreactive insulin derivatives, N epsilon B29-(azidobenzoyl) insulin (B29-MABI) and N alpha B1-(azidobenzoyl) insulin (B1-MABI), and were then exposed to light. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and radioautography of the crude plasma membrane fraction after reduction showed that B29-MABI labeled specifically three proteins of Mr 130 000, 90 000, and 40 000 whereas B1-MABI labeled specifically two proteins of Mr 130 000 and 40 000. B1-MABI also variably labeled some bands of intermediate Mr between 130 000 and 90 000. In contrast, the labeling of the 40-kilodalton protein was not observed in our previous studies in which photolabeling was carried our on isolated plasma membrane preparations [Yip, C. C., Yeung, C. W. T. & Moule, M. L. (1980) Biochemistry 19, 70-76; Yeung, C. W. T., Moule, M. L., & Yip, C. C. (1980) Biochemistry 19, 2196-2203]. Without reduction, an Mr 300 000 band and a larger band which barely entered a 5-15% gradient gel were specifically labeled by both photoreactive insulins. Reduction of these two high molecular weight bands gave rise to the 130-, 90-, and 40-kilodalton bands. The labeling of these proteins was affected neither by the time or temperature of incubation nor by the addition of methylamine, chloroquine, bacitracin, phenylmethanesulfonyl fluoride, p-(chloromercuri) benzenesulfonic acid, Trasylol, N-ethylmaleimide, or benzamidine. The labeling of these proteins by the photoreactive insulin derivatives was inhibited by first incubating the adipocytes with a human autoimmune serum to insulin receptor. We therefore conclude that these proteins are subunits of the insulin receptor in intact adipocytes.

Adipose Tissue↗
PubMed1982

Direct demonstration of insulin receptor internalization. A quantitative electron microscopic study of covalently bound 125I-photoreactive insulin incubated with isolated hepatocytes.

When 125I-insulin is incubated with isolated rodent hepatocytes at 37 degrees C, the ligand initially binds to the plasma membrane of the cell and is subsequently internalized by adsorptive endocytosis. To confirm directly that the insulin receptor is internalized with the ligand, we covalently linked photoreactive 125I-N sigma B29 (azidobenzoyl) insulin to its specific hepatocyte receptor and followed its fate by quantitative electron microscopic autoradiography. We found that the covalently linked photoreactive insulin is internalized by the cell in fashion analogous to the internalization of ordinary 125I-insulin, indicating that, at least under these conditions, the insulin receptor is internalized with the ligand.

Animals↗
PubMed1980

Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.

The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Affinity Labels↗