The biological and immunological properties of pork and beef insulin, proinsulin, and connecting peptides

Kitabchi, Abbas E.

doi:10.1172/jci106317

Cited by 73 publications

(29 citation statements)

References 17 publications

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“…This observation also appears to be true for proinsulin. Proinsulin is degraded by ISP at 1/30-1/50 the rate of insulin (11), while the binding of proinsulin to isolated cell membranes and the biological activity of proinsulin (23,24) are about 1/10 that of insulin (25).…”

Section: Discussionmentioning

confidence: 99%

Purification of Insulin-Specific Protease by Affinity Chromatography

Duckworth

Heinemann

Kitabchi

1972

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

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118

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A single enzyme that proteolytically degrades insulin was isolated from rat skeletal muscle. This enzyme was purified 1000-fold by a series of steps, including affinity chromatography on insulin bound to agarose at the NH2-terminal phenylalanine of the B chain. Insulin linked to agarose at the B-29 lysine residue did not bind the enzyme and, therefore, was not suitable for purification procedures. Insulin linked at the phenylalanine residue was a substrate for the enzyme and was degraded by it; insulin attached to agarose at the lysine residue was not degraded by the enzyme. The purified enzyme preparation yielded one major band on polyacrylamide gel electrophoresis, and elution of this area of the gel yielded insulin-degrading activity. The purified enzyme degraded insulin but not proinsulin, with a Km for insulin of 22 nM and a Ki for proinsulin of 40 nM. The enzyme is sulfhydryldependent, with a physiological pH optimum.

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Section: Discussionmentioning

confidence: 99%

Purification of Insulin-Specific Protease by Affinity Chromatography

Duckworth

Heinemann

Kitabchi

1972

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

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118

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Rat C peptide I and II stimulate glucose utilization in STZ-induced diabetic rats

Oshida

Kusunoki

et al. 1999

Diabetologia

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The C peptide of proinsulin has since its discovery been considered to be without biological effect of its own [1]. In recent years it has been reported, however, that C peptide increases muscle blood flow, glucose and oxygen uptake of the exercising forearm, decreases urinary albumin excretion and improves autonomic nerve function in patients with Type I (insulin-dependent) diabetes mellitus [2±6]. Moreover, C peptide has been shown to stimulate Na + ,K + -ATPase activity in renal tubular cells [7] and to activate the nitric oxide (NO) synthase of endothelial cells [8]. The rat has two proinsulins and thus two C peptides that differ with regard to two amino acids in the middle segment of the molecule [9]. Rat C peptide I and II are equipotent in their ability to stimulate Na + ,K + -ATPase [7]. Studies on isolated muscle strips from Type I diabetic patients and healthy subjects have confirmed the effect of C peptide on glucose transport in vivo and indicated that the stimulation occurs through pathways that do not include the insulin receptor [10]. C peptide does not, however, stimulate muscle glycogen synthesis in isolated mouse muscle [11]. Recent data show that glucose transport in rat muscle and adipose tissue can be Diabetologia (1999) AbstractAims. To study the effects of physiological concentrations of rat proinsulin C peptide I and II, respectively, on whole body glucose utilization in streptozotocin diabetic and healthy rats. Methods. A sequential insulin clamp procedure was used (insulin infusion rates 3.0 and 30.0 mU × kg ±1 × min ±1 ) in awake animals. C-peptide infusion rates were 0.05 and 0.5 nmol × kg ±1 × min ±1 . Blood glucose was clamped at 7.7 ± 0.3 mmol/l in the diabetic rats and at 3.9 ± 0.1 mmol/l in the healthy rats. Results. In diabetic rats infused at lower rates of C peptide and insulin, glucose utilization increased by 79±90 % (p < 0.001) compared with diabetic animals infused with saline and insulin. Increasing the rate of C-peptide infusion tenfold did not elicit a statistically significant further increase in glucose utilization. C peptide I and II exerted similar effects. The metabolic clearance rate for glucose in the diabetic animals infused with C peptide was not different from that of the healthy rats. During high-dose insulin infusion (30.0 mU´kg ±1´m in ±1 ) glucose utilization increased considerably and no statistically significant C-peptide effects were observed. About 85 % of the increase in glucose utilization induced by C peptide could be blocked by treatment with N-monomethyl-l-arginine. Conclusions/interpretation. Physiological concentrations of homologous C peptide stimulate whole body glucose utilization in diabetic but not in healthy rats. C peptide I and II elicit similar effects. The influence of C peptide on glucose utilization may be mediated by nitric oxide. [Diabetologia (1999) 42: 958±964]

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“…Demonstration of proinsulin as the precursor of insulin [5,6] with less biological activity [7,8] and suggestion of the presence of this prohormone and its intermediates in circulation [9, lo], prompted us to evaluate the relative suitability of these intermediates for insulin-degrading enzyme systems. The earliest work reported on the comparative ability of these enzymes in regard to substrate specificity was that of Brush [ll] in 1971, demonstrating that the supernatant fraction from a centrifugation of muscle homogenate a t 100000 x g can degrade insulin 30 times more than proinsulin.…”

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Substrate Studies for Insulin‐Specific Protease

Baskin

Kitabchi

1973

European Journal of Biochemistry

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Insulin-specific protease, a soluble cellular enzyme from rat skeletal muscle which has been purified recently as a single enzyme, has been studied in regard to its substrate specificity, using various immunoreactive and biologically active insulin and proinsulin intermediates. The rate of degradation of pork insulin taken as 1000/, was compared to other insulin and proinsulin derivatives. Porcine proinsulin intermediates consisting of cleaved proinsulin, desdipeptide, desnonapeptide and destridecapeptide-proinsulin, as well as desalanine, monoarginine and diarginine-insulin, were degraded at 19.8,25.6, 63.5,73.7, 101.5, 98 and 9S0/, of the activity of insulin, respectively.Rates of degradation of beef proinsulin, and intermediates I and IS were 6, 20. 8 Reduced proinsulin, labeled with iodo[14C]acetamide, did not show increased degradability by insulin-specific protease as compared to native proinsulin. These studies suggest that one requirement for optimal substrate activity may be the deblocking of the amino end of the A chain of insulin. The blocking of the amino end, which is present in proinsulin or proinsulin intermediate, will reduce degradability of these substrates by insulin-specific protease. The fact that the cleavage of disulfide bonds of proinsulin resulted in no further activation of proinsulin supports the above and indicates that steric hindrance may have a lesser role in the proinsulin molecule's inability to be degraded by insulin-specific protease.Two systems for degradation of insulin have been reported in the literature: 1. insulinase, a system proposed by Mirsky, which degrades insulin through the hydrolytic cleavage of the peptide bonds. This system was shown to have high specificity for insulin since it did not degrade other proteins such as albu- mined that the glutathione-dependent enzyme is located in the microsomes ; whereas, the glutathioneindependent enzyme is located in the cytosol fraction.Demonstration of proinsulin as the precursor of insulin its intermediates in circulation [9, lo], prompted us to evaluate the relative suitability of these intermediates for insulin-degrading enzyme systems. The earliest work reported on the comparative ability of these enzymes in regard to substrate specificity was that of Brush [ll] in 1971, demonstrating that the supernatant fraction from a centrifugation of muscle homogenate a t 100000 x g can degrade insulin 30 times more than proinsulin. This enzyme, which is called insulin-specific protease, has been demonstrated in all organs of rats [12] and has been purified in muscle [8], kidney [12] and liver [13] in our laboratories, showing that greater than goo/, degradation of insulin is accomplished with insulinspecific protease, not glutathione insulin transhydrogenase. Furthermore, with the use of affinity chromatography, insulin-specific protease has been purified from muscle of rat and has been shown by gel electrophoresis to be one single band. Insulinspecific protease is highly specific for insulin with a

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The biological and immunological properties of pork and beef insulin, proinsulin, and connecting peptides

Cited by 73 publications

References 17 publications

Purification of Insulin-Specific Protease by Affinity Chromatography

Purification of Insulin-Specific Protease by Affinity Chromatography

Rat C peptide I and II stimulate glucose utilization in STZ-induced diabetic rats

Substrate Studies for Insulin‐Specific Protease

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