Search PubMed⌕ Search

Biomedical subjects

R A Kohanski

Publications and source records attributed to R A Kohanski.

44 records · Page 3Linked to original sources
PubMed1986

Kinetic evidence for activating and non-activating components of autophosphorylation of the insulin receptor protein kinase.

Reduced and carboxamidomethylated-lysozyme (RCAM-lysozyme) is an excellent substrate (Km = 13 microM) and a potent inhibitor of receptor autophosphorylation (Ki = 0.6 microM). By using these properties of RCAM-lysozyme autophosphorylation was resolved into two kinetically and functionally distinct components involving formation of phosphotyrosine on the receptor's beta-subunits: 1. Insulin-stimulated autophosphorylation is independent of autophosphorylation at other sites; activation of insulin receptor-catalyzed substrate phosphorylation is dependent upon this component of autophosphorylation, which is inhibited by RCAM-lysozyme. 2. Autophosphorylation at saturating RCAM-lysozyme concentration is insensitive to insulin and has little effect on substrate phosphorylation. Thus, only insulin-dependent receptor autophosphorylation is responsible for activation of kinase-catalyzed substrate phosphorylation.

Catalysis↗
PubMed1985

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose.

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.

Adipose Tissue↗
PubMed1985

Posttranslational processing of the insulin proreceptor.

Following translation of the insulin proreceptor by 3T3-L1 adipocytes, about 1.5 hours is required for its conversion into active receptor; an additional 1.5 hours is needed for the active receptor to reach the plasma membrane. During this 3-hour period the proreceptor undergoes a complex series of processing events, glycosylation being an essential processing step. Thus, treatment of 3T3-L1 adipocytes with tunicamycin causes the depletion of cellular insulin binding activity and the accumulation of an inactive aglyco proreceptor. To investigate posttranslational processing of normal proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first 35S-labeled intermediate detected is a 190-kDa polypeptide (proreceptor) which is rapidly (t1/2 = 15 minutes) processed into a 210-kDa species. Both polypeptides contain N-linked core oligosaccharide chains, but in the latter case these chains appear to contain terminal N-acetylglucosamine. The 210-kDa precursor is converted slowly (t1/2 = 2 hours) by proteolytic processing into a 125-kDa (alpha') and 83-kDa (beta') species. Immediately prior to insertion into the plasma membrane, 3 hours after its synthesis, the alpha' and beta' precursors are converted to mature receptor composed of alpha (135 kDa) and beta (95 kDa) subunits. The 125-kDa alpha' and 83-kDa beta' precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the alpha' and beta' precursors are sialylated, giving rise to the 135-kDa alpha and 95-kDa beta receptor subunits and becoming Endo H resistant and neuraminidase sensitive. In the presence of tunicamycin, a 180-kDa aglyco receptor polypeptide accumulates which is not further processed and does not reach the cell surface. It is concluded that N-linked oligosaccharide chains on the proreceptor are required either for its intracellular translocation to the proteolytic cleavage site or for its identification as a target of the cleavage enzyme. Thus, glycosylation of the insulin proreceptor is crucial for proper processing and formation of functional receptor.

Cell Membrane↗
PubMed1985

Receptor affinity chromatography.

Insulin receptor was purified in high yield from cultured 3T3-L1 mouse adipocytes using the bifunctional ligand N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin in conjunction with avidin-Sepharose CL-4B. This ligand is 100% competent as insulin and 60% competent as biotin, as measured by competitive binding assays. The procedure requires preliminary removal of biotin-containing proteins on "native" avidin-Sepharose CL-4B. This matrix shows nearly the same biotin-binding characteristics as uncoupled avidin and can be regenerated by washing with 0.02 N HCl, causing only a minor loss of nonexchangeable biotin-binding sites. Insulin receptor is isolated by formation of a complex between the bifunctional ligand and the receptor, and then adsorption to "monomeric" avidin-Sepharose via the biotin moiety. This affinity matrix binds [14C]biotin with a Kd approximately equal to 0.2 microM and has exchangeable/nonexchangeable biotin binding sites in the ratio 9:1. Displacement of homogeneous insulin receptor is achieved by the addition of biotin; the elution is time-dependent, suggesting that it is accomplished by the prevention of rebinding.

Adipose Tissue↗
PubMed1984

Role of glycosylation in the processing of newly translated insulin proreceptor in 3T3-L1 adipocytes.

A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [35S]methionine after which 35S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first 35S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t1/2 approximately equal to 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t1/2 approximately equal to 2 h) proteolytically processed to 125-kDa (alpha') and 83-kDa (beta') precursors of the mature alpha- and beta-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the alpha'- and beta'-precursor polypeptides were converted to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa alpha'- and 83-kDa beta'-species, and the mature alpha- and beta-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-kDa alpha'- and 83-kDa beta'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the alpha'- and beta'-species by sialic acid occurs, giving rise to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and proteolytic cleavage to the alpha- and beta (or alpha'- and beta'-)-subunits. The 180-kDa species, which appears to be the aglyco-form of hte 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin re

Adipose Tissue↗
PubMed1983

Binding of insulin to solubilized insulin receptor from human placenta. Evidence for a single class of noninteracting binding sites.

Binding of 125I-insulin to insulin receptor was studied at equilibrium using plasma membrane protein, from human placenta, solubilized in Triton X-100. Measured at 1 nM 125I-insulin, the amount of radioligand bound specifically was dependent upon pH, the optimal association at 4 degrees C occurring in the pH range 8.2-8.7. The Adair equation was employed for analysis of binding isotherms generated at several pH values for concentrations of radioligand varying from 0.01-8.0 nM. Association constants derived from these analyses showed the same pH dependence described above and were found to be independent of receptor concentration. Hill plots derived from these titrations consistently yielded Hill coefficients of 1 and Scatchard plots gave virtually straight lines. Close correspondence was found between theoretical analyses and observations under all experimental conditions and graphical methods employed. These results are consistent with a single class of noninteracting 125I-insulin binding sites on the solubilized insulin receptor.

Binding Sites↗
PubMedDate not supplied

Post-translational processing and activation of insulin and EGF proreceptors.

We have investigated the role of glycosylation on the post-translational processing of the insulin, and EGF proreceptor polypeptides. Following translation of the insulin proreceptor, by 3T3-L1 adipocytes, about 1.5 h are required for its conversion into active receptor; an additional 1.5 h are needed for the active receptor to reach the plasma membrane. During this 3-hour period the proreceptor undergoes a complex series of processing events, glycosylation being an essential processing step. Thus, treatment of 3T3-L1 adipocytes with tunicamycin caused the loss of cellular insulin binding activity and the accumulation of an inactive aglyco-proreceptor. Similarly, it was demonstrated in human A431 epidermoid carcinoma cells that the initial EGF-proreceptor (160 kDa) translation product undergoes a slow (t 1/2 = 30 min) processing step by which ligand (EGF) binding activity was acquired. It was shown that N-linked core oligosaccharide addition is essential for this critical processing step and the acquisition of EGF binding activity. This was found not to require the conversion of high mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed. To investigate post-translational processing of normal insulin proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first 35S-methionine-labeled intermediate detected is a 190 kDa polypeptide (proreceptor) which is rapidly (t 1/2 = 15 min) processed into a 210 kDa species. Both polypeptides contain N-linked core oligosaccharide chains, but in the latter case these chains appear to contain terminal N-acetylglucosamine. The 210 kDa precursor is converted slowly (t 1/2 = 2 h) by proteolytic processing into a 125 kDa (alpha') and 83 kDa (beta') species. Immediately prior to insertion into the plasma membrane, 3 h after its synthesis, the alpha' and beta' precursors are converted to mature receptor comprised of alpha-(135 kDa) and beta-(95 kDa) subunits. The 125 kDa alpha'- and 83 kDa beta'-subunit precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the alpha' and beta' precursors are sialylated, apparently in the Golgi apparatus, giving rise to the 135 kDa alpha and 95 kDa beta receptor subunits and become Endo H-resistant and neuraminidase-sensitive. A proposed sequence of post-translational processing events for the insulin proreceptor is shown in Figure 10.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylglucosaminidase↗