[LeuB24]insulin and [AlaB24]insulin: altered structures and cellular processing of B24-substituted insulin analogs.

Abstract

We have used insulin analogs having leucine or alanine substitutions at positions B24 and B25 to examine the structural basis for insulin binding and insulin metabolism by isolated rat hepatocytes. Apparent receptor binding affinities for the analogs were in the order insulin > [LeuB24]insulin > [LeuB25insulin = [AlaBU]insulin. Incubation of the corresponding "2I-labeled peptides with hepatocytes followed by analysis of the cell-associated products showed that ['251] [MaBlB]insulin (all derivatives of the porcine hormone) were prepared essentially as described (8). The final preparations gave the expected amino acid compositions after hydrolysis in 6 M HCl for 20 hr at 110°C and were shown to contain <10% monodesamido and mono-protected forms by polyacrylamide gel electrophoresis at pH 8.7 (21).Radioiodination of Insulin and Insulin Analogs. Radiolabeling methods were based on the procedure of Freychet et al. (22) but were modified to restrict the extent ofiodination to 0.2-0.3 mol of "I per mol ofpeptide. Carrier-free Nalm2I (1 nmol, about 5 ,ul, Industrial Nuclear, St. Louis) was diluted with 20 ,ud of 1.5 M potassium phosphate buffer at pH 7.5; fresh chloramine-T (2 nmol in 1 ,ud of 30 mM sodium phosphate buffer, pH 7.5) was then added and the solution was allowed to stand at 22°C for 10 sec. The mixture was diluted with 50 ,l of the sodium phosphate buffer and the insulin or insulin analog (3 nmol in 5 ,ul of0.75 M acetic acid) was added with mixing. After 10 min, the iodination was stopped by the addition of N-acetyl-L-tyrosine (1 ,umol in 20 ,ul of the sodium phosphate buffer) and the radiolabeled peptide was purified by adsorption to a column of Whatman CF-il cellulose (23) and elution with 12.5% (wt/vol) bovine serum albumin (fraction V, Miles) in 30 mM sodium phosphate buffer at pH 7.5.Analysis of the positions of label in the radioiodinated peptides proceeded from either oxidative sulfitolysis or digestion with Staphylococcus aureus protease prior to oxidative sulfitolysis (20). Samples were subjected to paper electrophoresis in 30% (wt/vol) formic acid. The Pauly reagent was used for identifying tyrosine and histidine residues, phenanthrenequinone for identifying arginine residues (24), and autoradiography for detecting radiolabeled peptides.Hepatocyte Isolation and Incubation. Rat hepatocytes were isolated by collagenase digestion and were incubated with radiolabeled and unlabeled insulin as described (1,20 The publication costs ofthis article were defrayed~in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Conformational basis of the receptor-binding potency of normal and mutant insulin molecules with relevance to the pathophysiology of noninsulin dependent diabetes mellitus (NIDDM)

“…These are reasonably close to the values of $*, $J~, q3, +4, and $4 obtained in our study (see Table I). Secondly, our results are consistent with the observation [21] that residue B25 appears on the face of the insulin molecule and B24 protrudes into the molecule (see Fig. 2).…”

Identification of a mutant human insulin predicted to contain a serine-for-phenylalanine substitution.

“…As the relative hydrophobicities of the side-chain replacements play little role in determining the activities of the final products, the loss of a phenylalanyl side chain appears to be the specific cause of decreased activity in each case. For analogs with replacements at position B25, decreased activity likely results from the loss of a specific side-chain contact with the receptor (perhaps involving iW-orbital interactions between hormone and receptor aromatic ring systems); for analogs with replacements at position B24, decreased activity is probably due to conformational changes in the insulin monomer resulting from altered side-chain bulk, rather than from altered side-chain functionality (6,8,11,17 …”

Binding of quinine- and quinidine-dependent drug antibodies to platelets is mediated by the Fab domain of the immunoglobulin G and is not Fc dependent.

“…Each batch ofplatelet membranes was evaluated using radioimmunoassay (RIA) for PlAI antigen (22). Specifically, glycoprotein IlIa was purified by differential detergent extraction (23) and gel filtration on Sephacryl S-300 (24) and radiolabeled using a chloramine-T method (25). The immunoassay consisted of mixing this radiolabeled antigen with anti-PI" at molar equivalence and precipitating the resulting antigen-antibody complexes with formalin-fixed S. aureus to obtain the maximum value for percent binding in Fig.…”