Syntheses of biotinylated and dethiobiotinylated insulins

Zhang, Wei Jun; Romovacek, Hana; Finn, Frances M.; Bothner‐By, Aksel A.; Mishra, Panchanand

doi:10.1021/bi00307a002

Cited by 23 publications

(11 citation statements)

References 16 publications

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“…To discover such derivatives, we synthesized a series of biotinyl-X insulins (7,21), in which the length of the spacer X was systematically increased, and we measured the rate of dissociation of their complexes with succinoylavidin. The t112 for dissociation of the biotin succinoylavidin complex is 127 days (20).…”

Section: I-labeled Receptor Is Shown Inmentioning

confidence: 99%

“…It could be argued that the affinity resin binds the receptor via hydrophobic interactions, because it contains a long stretch of aliphatic hydrocarbon chain. To eliminate this possibility, we conducted experiments with columns containing (6-biotinylamido)hexanoic acid (7) attached to the Sepharose 4B immobilized succinoylavidin and found that they failed to bind significant amounts of receptor (experiments not shown).…”

Section: I-labeled Receptor Is Shown Inmentioning

confidence: 99%

See 1 more Smart Citation

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

Finn¹,

Titus²,

Horstman³

et al. 1984

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

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The ligand NB'-(6-biotinylamido)hexanoylinsulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, (iii) wheat germ agglutinin purification, and (iv) insulin-affinity chromatography. NaDodSO4/PAGE of the purified '2I-labeled receptor under nonreducing conditions showed the presence of a major component with an approximate molecular weight of 350,000 and a minor component with a molecular weight of =166,000. Based on the assumption that the degree of labeling is comparable in both components, the material corresponding to the Mr 350,000 peak represents =94% of the receptor preparation as determined by scanning the autoradiograms. The specific insulin binding capacity of the preparation is 18 ± 6 pbg of 12'I-labeled insulin per mg of protein as determined by the polyethylene glycol assay and analyzed by Scatchard plot. Insulin binding activity was stable at 40C and pH 7.6 for at least 12 weeks but was destroyed by freezing and thawing. The availability of highly purified receptor afforded the opportunity to explore its precipitability by polyethylene glycol under assay conditions. Whereas trichloroacetic acid precipitated 95% of the '5I-labeled receptor, polyethylene glycol precipitated only 30%. If the specific activity of the receptor is corrected for incomplete precipitability by polyethylene glycol, the apparent specific binding would be 3.5 + 1.2 mol of insulin per mol of receptor. These results are in disagreement with the current receptor model, which postulates that 1 mol of receptor (Mr, 350,000) binds 2 mol of insulin. Clearly, the problems associated with the method available for determining insulin binding are sufficiently serious to preclude their use in determining receptor valence.Affinity chromatography for insulin receptor isolation was introduced by Cuatrecasas and his colleagues in 1972 (1, 2). An affinity resin was prepared by attaching unprotected insulin to Sepharose via a spacer arm. Using this resin, Jacobs et al. (3) were able to isolate a crude insulin receptor preparation from Triton X-100 solubilized rat liver membranes. The same affinity resin was used by Siegel et al. (4) for the retrieval of insulin receptor preparations from human placenta. The receptor was eluted from the affinity resin with 4.5 M urea (pH 6.0), conditions that damage the receptor. Recently, Fujita-Yamaguchi et al. (5) observed that insulin receptors with high specific insulin binding activity can be eluted from Cuatrecasas-type columns with acetate buffer, pH 5.0/1 M sodium chloride. A considerable store of information concerning the nature of insulin receptors has been accumulated by indirect methods using tracer amounts of materials (6), but methodology for isolating large quantities of receptor is not available. Finite information regarding t...

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Section: I-labeled Receptor Is Shown Inmentioning

confidence: 99%

Section: I-labeled Receptor Is Shown Inmentioning

confidence: 99%

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

Finn¹,

Titus²,

Horstman³

et al. 1984

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

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“…Pepstatin is a potent transitionstate analogue inhibitor of aspartic proteases, which can be strongly bound in the active-site cleft. The binding of substrate as deduced from the binding of pepstatin and a pepstatin analogue provides the clue as to how to place the scissile peptide bond with respect to the catalytic groups, Asp 32 and Asp 215 [8,15,18,19]. This binding mode indicates that it is Asp 32 (pepsin numbering) that catalyses the nucleophilic attack by the water molecule.…”

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confidence: 99%

The mechanism of action of aspartic proteases involves ‘push‐pull’ catalysis

Polgár

1987

FEBS Letters

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In accord with the available kinetic and X-ray crystallographic data, it is proposed that the two catalytically competent carboxyl groups of aspartic proteases constitute a functional unit which mediates the proton from the attacking water molecule to the leaving nitrogen atom of the substrate. Protonation of this nitrogen atom has been the main issue of the previous mechanistic proposals. The first step of the present mechanism involves proton transfer from the water to the aspartic diad and concurrently another proton transfer from the diad to the carbonyl oxygen of the scissile peptide bond. These proton transfers provide the driving force for the bond formation between the substrate and water, which leads to the formation of a tetrahedral intermediate. The intermediate breaks down to products by a similar facilitation, i.e. by concerted general acid-base catalysis, which involves simultaneous proton transfers from the intermediate to the diad and from the diad to the leaving nitrogen of the substrate. The symmetrical mechanism of the formation and decomposition of the tetrahedral adduct resembles that found in the serine protease catalysis.Pepsin; Aspartic protease; Enzyme mechanism Aspartic proteases include several important enzymes, such as pepsin, chymosin, renin, cathepsin D and the proteases isolated from numerous fungi. The amino acid sequences of all these enzymes are homologous, in particular around the active-site residues. Comprehensive reviews on aspartic proteases have been published [1][2][3]. The threedimensional structure of pepsin [4] and three microbial aspartic proteases including penicillopepsin [5,6], Rhizopus chinensin protease [7] and Endothiaparasitica protease [7] has been reported. The tertiary structures of pepsin and the microbial Correspondence address: L. Polg~ir,

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“…Found: C, 53.30; H, 5.67; N,8.49;Cl,caproyl]amino]-4-chloro-3-(2phenylethoxy)isocoumarin (BIC 3). 6-(Biotinylamino)caproic acid was prepared from the JV-hydroxysuccinimide ester of biotin (Jasiewicz et al, 1976) and methyl 6-aminocaproate acid hydrochloride by a previously described method (Hofmann et al, 1984). ¿-(Biotinylamino)caproic acid chloride was prepared by incubating 6-(biotinylamino)caproic acid (0.36 g, 1 mmol) in 4 mL of thionyl chloride at 25-35 °C for 1 h and removing excess thionyl chloride in vacuo.…”

Section: Methodsmentioning

confidence: 99%

Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases

Kam

Abuelyaman²,

Li³

et al. 1993

Bioconjugate Chem.

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Eight new biotinylated, mechanism-based isocoumarin serine protease inhibitors have been designed and synthesized to detect, localize, and isolate serine proteases. Isocoumarins that contain a 4-chloro group, a biotinylated substituent at the 7-position, and different 3-alkoxy groups are inhibitors of various serine proteases including human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), trypsin, human recombinant granzyme A, chymotrypsin, and cathepsin G. Insertion of spacers between the isocoumarin moiety and the biotin moiety enhanced enzyme inhibitory potency and may also promote binding of the enzyme-inhibitor complex to avidin. The 3-alkoxy groups conferred selectivity toward different serine proteases with chymotrypsin being inhibited effectively by compounds with 3-phenylethoxy groups while derivatives with 3-methoxy, ethoxy, or propoxy groups were potent inhibitors of HLE and moderate inhibitors of PPE. Full enzymatic activity was regained after the immediate addition of hydroxylamine to the inactivated chymotrypsin and PPE derivatives, which indicated that a simple acyl enzyme derivative is formed initially in the inhibition reaction. Egg avidin did not effect the rate of spontaneous enzyme reactivation rate while streptavidin accelerated the reactivation reaction. PPE inhibited by 7-[[6-(biotinylamino)caproyl]amino]-4-chloro-3- ethoxyisocoumarin (BIC 5) or 7-[[6-[[6-(biotinylamino)caproyl]amino] caproyl]amino]-4-chloro-3-methoxyisocoumarin (BIC 7) was bound to immobilized avidin columns. Most of inhibited PPE could be eluted from the monomeric or tetrameric avidin columns but only a portion (40-70%) of the enzyme was active due to the partial formation of a stable alkylated enzyme derivative during the isolation process.(ABSTRACT TRUNCATED AT 250 WORDS)

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Syntheses of biotinylated and dethiobiotinylated insulins

Cited by 23 publications

References 16 publications

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

The mechanism of action of aspartic proteases involves ‘push‐pull’ catalysis

Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases

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