Wondering

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Shaymaa Ahmed
Shaymaa  Ahmed's picture
Wondering

Hi

I've been searching for the rat insulin ELISA kit and found that there is an ultrasensitive Rat/human ELISA kit. Does this mean that the antibody is directed against a common shared part in rat and human isulin and if this is right can any body send me the difference in structue between rat and human isulin I'll be grateful.

Thanks

Tony Rook
Tony Rook's picture
Here is the structure for

Here is the structure for human insulin as reported here.

And here is a R6 Human Insulin Hexamer (Symmetric), Nmr, 'red' Substate, Average Structure as reported here.

Unfortunately, I was not able to quickly locate the structure of rat insulin. If anybody else has this information please post.

But here are a few references that may help you out...

Gregor Majdic, Allan S. McNeilly, Richard M. Sharpe, Lee R. Evans, Nigel P. Groome, and Philippa T. K. Saunders. Testicular Expression of Inhibin and Activin Subunits and Follistatin in the Rat and Human Fetus and Neonate and During Postnatal Development in the Rat. Endocrinology, May 1997; 138: 2136 - 2147.

Inhibins, activins, and follistatins are all believed to play roles in
the regulation of FSH secretion by the pituitary and in the paracrine regulation of testis function. Previous studies have resulted in conflicting data on the pattern of expression of the inhibin/activin subunits, and little information on expression of follistatin during fetal/neonatal life. We have made use of new, highly specific monoclonal antibodies and fixed tissue sections from fetal, neonatal, and adult rats, and limited amounts of fetal and neonatal human testis, to undertake a detailed immunocytochemical study of the pattern of expression of these regulatory proteins.

In the rat, positive immunostaining for the a-subunit of inhibin (a) was first detectable on day 14.5 post coitum (p.c.), the first day on which the testis could be morphologically distinguished from the ovary. During fetal life, the a-immunostaining was most prominent in the fetal Leydig cells. In Sertoli cells, a-immunostaining was slightly stronger on days 14.5 and 15.5 p.c. compared with 16.520.5. After birth, a-immunostaining remained intense in fetal Leydig cells but declined following their replacement with their adult-type counterparts; in contrast, a-subunit increased in Sertoli cells immediately after birth. Immunostaining with antibodies specific to bB-subunit showed a similar pattern to that of the a-subunit, except that positive
immunostaining was first detectable on day 16.5 p.c., 2 days later than immunostaining for the a-subunit. The pattern of bB-immunostaining in postnatal samples paralleled that of the a-subunit. Immunostaining using antibodies against the bA-subunit did not produce any significant reaction product in any sample. Follistatin was undetectable in the fetal rat testis but appeared in the Leydig cells immediately after birth and its expression remained intense throughout postnatal development and in adult testis. No evidence was obtained for expression of either the inhibin/activin subunits or follistatin in the germ cells, peritubular myoid cells, or other interstitial cells in any of the sections examined. In the human fetal testis, both a- and
bB-subunits were immunodetectable at 16, 18, and 24 weeks gestation in Sertoli and Leydig cells, with stronger immunostaining in Sertoli cells at 24 weeks. Postnatally at 4 months, immunoexpression of the bB-subunit was no longer detectable, whereas the a-immunostaining became weaker but was still present in both Sertoli and Leydig cells. No positive immunostaining for bA-subunit or follistatin was detectable at any time point studied.

In conclusion, we have shown that, in the rat testis, the majority
of inhibin a-subunit and inhibin/activin bB-subunit is immunolocalized to the fetal-type Leydig cells during fetal/neonatal life but, following birth, immunoexpression in the Sertoli cells of both subunits increases markedly while follistatin is immunodetectable only postnatally.

BJ Goldstein and AL Dudley. The rat insulin receptor: primary structure and conservation of tissue- specific alternative messenger RNA splicing. Molecular Endocrinology, Vol 4, 235-244.

Abstract:
To investigate whether alterations in the polypeptide structure of the insulin receptor might explain the heterogeneity observed in its properties between species and in different tissues, we obtained the complete primary structure of the rat liver insulin receptor precursor by cDNA cloning and sequencing. The rat proreceptor contains 1357 amino acids and has 95.2% identity with deduced polypeptide sequences reported for the human insulin receptor precursor. In addition, the rat liver insulin receptor cDNA was similar to the form of the human insulin receptor mRNA that contains Exon 11 in its coding region, which undergoes tissue-specific alternative splicing. Using the polymerase chain reaction to amplify a cDNA segment corresponding to this region in several rat tissues, the splicing pattern of sequences homologous to Exon 11 was found to be highly conserved, providing further evidence that these two forms of the insulin receptor may serve important functional or regulatory roles. These studies have also identified subtle variations in the primary structure of the insulin receptor which may influence its properties between species.

JOHN B. SCHWEITZER, ROBERT M. SMITH, AND LEONARD JARETT. Differences in organizational structure of insulin receptor on ratadipocyte and liver plasma membranes: Role of disulfide bonds. Proc. Natl. Acad. Sci. USA Biochemistry. Vol. 77, No. 8, pp. 4692-4696, August 1980

Abstract:
Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times contro values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0 X 108 M-l which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 1251-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented
oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol.
Lve lasma membranes treated with up to 1 mM dithiothreitol exhiitda maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

Tony Rook
Tony Rook's picture
Here's another reference that

Here's another reference that may be of some interest...

RICHARD A. ROTH, DELANIE J. CASSELL, K. Y. WONG, BETTY A. MADDUX, AND IRA D. GOLDFINE. Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action. Proc. Nati Acad. Sci. USA Cell Biology
Vol. 79, pp. 7312-7316, December 1982.

Abstract:
Antibodies to the insulin receptor were prepared
in BALB/c mice by immunization with IM-9 human lymphocytes,
a cell type that has a large number of plasma membrane insulin
receptors. The spleens ofthese mice-were then removed, and theirlymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgGI antibodies directed towards the insulin receptor. This antibody blocked 1251-labeled insulin binding to its receptor by more than 90% in three human tissues: IM-9 cultured lymphocytes, freshly isolated adipocytes, and placenta membranes. In contrast, the antibody did not inhibit insulin binding to rat adipocytes and rat liver plasma membranes, suggesting that the antibody was species specific. In IM-9 cells, which had their proteins prelabeled with [PS]methionine, the antibody precipitated two polypeptides with molecular weights of 135,000 and 95,O00; these molecular weights are identical to those previously identified as the a and fi subunits of the insulin receptor. The monoclonal antibody inhibited the actions of insulin on both human adipocytes and fibroblasts, suggesting that the antibody was an antagonist of insulin action. The present studies suggest, therefore, that monoclonal antibodies to the insulin receptor may provide
new insights into the structure of the insulin receptor and its
interaction with insulin.