Preparation of Crystalline Insulin

Romans, R. G.; Scott, Darren M.; Fisher, A. M.

doi:10.1021/ie50367a010

Cited by 41 publications

(12 citation statements)

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“…This application of an acid/ethanol extraction procedure has been demonstrated to be useful for extracting transforming proteins from samples of 10-130 g of tissue and can easily be scaled up to the processing of kg quantities of starting materials (21). It satisfies the requirements for minimizing proteolytic activity and can extract acid-stable proteins in high yield, as evidenced by the report of recoveries of insulin from the pancreas of greater than 90% with this method (3).…”

Section: Discussionmentioning

confidence: 99%

Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction.

Roberts¹,

Lamb²,

Newton³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

377

201

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Polptides characterized by their ability to confer a transformed phenotype on an untransformed indicator cell have been isolated directly from tumor cells growing both in culture and in the animal, by using an acid/ethanol extraction procedure. Assay of these polypeptides is based on their ability to induce normal rat kidney fibroblasts to form colonies in soft agar. Peptides from murine sarcoma virus-transformed mouse 3T3 cells grown in culture had the highest specific activity in this assay; peptides from sarcomas produced from these cells or from chemically induced transplantable bladder carcinomas of mice were on d as active; and peptides from a chemically induced rat tracheal carcinoma had only one-tenth the activity. Treatment with either trypsin or dithiothreitol destroyed the activity of all of these materials. The properties of these intracellular polypeptides from both virally and chemically transformed cells are similar to those described for the sarcoma growth factors (SGFs) previously isolated from the conditioned medium of sarcoma virus-transformed mouse 3T3 For the purposes of purification of these polypeptides, it was desired to extract them directly from tumor cells, rather than from conditioned medium. It was also important to ascertain whether the SGFs were unique to MuSV transformation, or whether they were representative of a class of proteins with similar properties, which we shall call transforming growth factors (TGFs) (2). We now report finding TGFs in cultured MuSV-transformed 3T3 cells and in sarcomas produced by inoculation of these cells into athymic mice. Moreover, TGFs of specific activity approximately equal to that of the sarcomas have been isolated from chemically induced bladder carcinomas. These TGFs have been extracted from the cells with acid/ethanol, a procedure that has previously been applied to the extraction from organs and from blood of such biologically active polypeptides as insulin (3, 4), glucagon (5), insulin-like growth factor (6), the somatomedins (7), and secretin (8). The demonstrated application of this method to the finding of a class of acid-stable TGFs from cells transformed by either a virus or chemicals brings a unifying concept to the mechanism of carcinogenesis. MATERIALS AND METHODSMuSV-Transformed 3T3 Cells. The Moloney MuSV-transformed 3T3 cell line 3B11-1C was grown in roller bottles as described (1). After the removal of the "sarcoma-conditioned medium," cells were scraped from the bottles into phosphatebuffered saline. After centrifugation at 500 X g for 10 min, the supernatant was discarded and the remaining cell pellet was frozen in the gas phase of a liquid nitrogen freezer.Sarcomas Derived from MuSV-Transformed 3T3 Cells. Cells of the Moloney MuSV-transformed 3T3 cell line 3197-3 (9), deficient in leukemia helper virus, were scraped into sterile phosphate-buffered saline, and 1 X 106 cells were inoculated subcutaneously into nude mice. Tumors were harvested at 2 weeks to 2 months and immediately frozen and stored above liquid nitrog...

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Section: Discussionmentioning

confidence: 99%

Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction.

Roberts¹,

Lamb²,

Newton³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

377

201

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“…1>2>17>18 Crystalline rabbit insulin was not available for neu- tralization tests and crude extracts of rabbit pancreas were prepared by extraction with a salt-alcohol mixture as described by Romans. 13 Beef pancreatic extracts prepared by this technic had hypoglycemic activity that was completely antagonized in immunized rabbits (figure 5A). In contrast, different preparations obtained from rabbit pancreas were neutralized to varying extents ( figure 5B).…”

Section: Detection Of Antibodies To Insulin With Liver Extractmentioning

confidence: 94%

The Reaction of Rabbit Anti-beef Insulin Serum with Heterologous and Homologous Insulin Preparations

Narahara

Williams

1964

Diabetes

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A method for assaying antibodies to insulin that is based on the protection of insulin-I-131 from degradation by a liver extract is described. Moderately potent antibodies to insulin were obtained in rabbits by the injection of crystalline beef insulin, and a high degree of insulin resistance was demonstrable in some of the animals. The immunized animals did not become diabetic.The biological activity of various insulin preparations was detected by their ability to lower the blood sugar of rabbits or through the stimulation of glucose uptake by the rat hemidiaphragm. Crystalline beef insulin and crude extracts of beef pancreas were effectively neutralized under the conditions of the tests whereas corresponding pork insulin preparations were incompletely neutralized in the rat hemidiaphragm assay. The insulin activity of sera from beef, pork, sheep and human sources was neutralized by the rabbit antisera.The insulin activity of rabbit pancreas extracts and rabbit sera exhibited significant individual differences in the extent of neutralization. These observations on rabbit insulin preparations support the concept of Moloney and Coval that insulin of a given animal species can exist either in a "native" form that is not readily neutralized by antiserum produced in the same species, or an "altered" form that can be neutralized.

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“…The frozen pancreases were extracted by either the acid-alcohol procedure (4,5) or the salt-alcohol procedure described by Romans (7,8).…”

Section: Methodsmentioning

confidence: 99%

“…Procedures for the isolation and crystallization of insulin from the pancreas of various species on a commercial scale are well-known (4)(5)(6)(7)(8). Repeated attempts by us and others (9), however, to apply these procedures, on a laboratory scale, to the human pancreas proved unsuccessful.…”

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confidence: 99%