Preparation and characterization of novel substrates of insulin proteinase (EC 3.4.99.45).
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ABSTRACT:
SUBMITTER: Werlen RC
PROVIDER: S-EPMC1137316 | biostudies-other | 1994 Sep
REPOSITORIES: biostudies-other
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Project description:We have studied a major product in the degradation of insulin by insulin proteinase (EC 3.4.22.11). Semisynthetic [[3H]PheB1]insulin and [[3H]GlyA1]insulin were used in the experiments. The structure of the fragment was deduced by observing the chromatographic and electrophoretic migration of the label both before and after further digestion of the fragment with proteinases of known specificity, with and without additional treatment by performic acid. Ambiguities were resolved by studying the behaviour of authentic fragments of known structure, isolated and characterized after digestion of intact insulin by proteinases of known specificity. We conclude that a major product in the degradation of insulin by insulin proteinase consists of a truncated section of the A chain, joined by the disulphide bridge B7-A7 to a truncated section of the B chain. The A-chain fragment consists most probably of residues A1-A13, and the B-chain fragment consists most probably of residues B1-B9. The similarity between this fragment and that found by other workers when insulin is degraded by intact hepatocytes is significant in the light of proposals that insulin proteinase is a possible participant in the physiological degradation of insulin by target cells.
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2022-08-02 | GSE210124 | GEO
Project description:Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin-agarose in a reversible manner. The K(d) values for factor VIII, fibrinogen, factor XIII and protein C were 1.2x10(-8), 4.4x10(-8), 2.8x10(-7) and 8.1x10(-5) M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (k(ass)) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.
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Project description:Purpose: With the increase of population aging and the proportion of overweight and obese, a growing number of people are suffering from diabetes. Insulin (INS) as the most widely used hypoglycemic agent was always chosen as the most effective treatment method of diabetes. In this study, fumaryl diketopiperazine (FDKP) was used as a carrier for the pulmonary delivery of insulin. Patients and methods: The INS-loaded FDKP microspheres (INS@FDKP-MPs) were prepared by spray drying and physicochemical properties (drug loading, particle size, flowability, moisture content, morphology, and crystalline state) were further investigated. Pharmacodynamics was investigated on diabetic model rats administrated by intratracheal insufflation. Results: The INS-loaded FDKP microspheres show satisfied flowability and in vitro deposition with FPF 50.2% and MMAD 3.45 ± 0.13 μm, and the blood glucose level was significantly decreased. Moreover, no inflammatory reaction was observed during the safety study. Conclusion: To sum up, the aim was to develop a non-injection system for insulin, INS@FDKP-MPs powder inhalation with high dose, low toxicity, and good lung deposition inhalation could rapidly decrease the blood glucose level without immune stimulation, which shows remarkably potential on diabetes treatment by pulmonary delivery route.
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Project description:Solid-phase synthesis was used to prepare a series of modifications to the selective and potent inhibitor of endopeptidase EC 3.4.24.15 (EP24.15), N-[1(R, S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), which is degraded at the Ala-Tyr bond, thus severely limiting its utility in vivo. Reducing the amide bond between the Ala and Tyr decreased the potency of the inhibitor to 1/1000. However, the replacement of the second alanine residue immediately adjacent to the tyrosine with alpha-aminoisobutyric acid gave a compound (JA-2) that was equipotent with cFP, with a K(i) of 23 nM. Like cFP, JA-2 inhibited the closely related endopeptidase EC 3.4.24.16 1/20 to 1/30 as potently as it did EP24.15, and did not inhibit the other thermolysin-like endopeptidases angiotensin-converting enzyme, endothelin-converting enzyme and neutral endopeptidase. The biological stability of JA-2 was investigated by incubation with a number of membrane and soluble sheep tissue extracts. In contrast with cFP, JA-2 remained intact after 48 h of incubation with all tissues examined. Further modifications to the JA-2 compound failed to improve the potency of this inhibitor. Hence JA-2 is a potent, EP24.15-preferential and biologically stable inhibitor, therefore providing a valuable tool for further assessing the biological functions of EP24.15.
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