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M Fabry

Publications and source records attributed to M Fabry.

29 records · Page 2Linked to original sources
PubMed1993

Heterogeneity of insulin receptors in rat tissues as detected with the partial agonist B29,B29'-suberoyl-insulin.

Using the insulin receptor partial agonist B29,B29'-suberoyl-insulin, a covalently dimerized insulin derivative, we previously demonstrated a heterogeneity of signal transduction by insulin receptors in two cell systems. The present study was designed to characterize the heterogeneity of insulin receptors in different rat tissues with this agent. Binding of 125I-insulin to insulin receptors and its inhibition by B29,B29'-suberoyl-insulin or by unlabeled insulin were assayed in plasma membranes from brain, spleen, adipocytes, and liver. IC50 values of B29,B29'-suberoyl-insulin were different in all tissues investigated (brain < spleen < adipocytes < liver). In contrast, IC50 values of insulin were identical, with the exception of spleen membranes (spleen < brain = adipocytes = liver). Furthermore, the IC50 ratios (B29 dimer/insulin) were significantly different, ranging from 0.7 (brain) to 12.8 (liver). Solubilization and partial purification of insulin receptors failed to abolish the marked difference between brain and liver (IC50 ratios of 1.8 and 7.1, respectively). The apparent molecular masses of the alpha subunits of insulin receptors, as labeled with a photoreactive insulin derivative, appeared identical in liver and spleen but were significantly lower in adipocytes and brain (liver = spleen > adipocytes > brain). The tissue-specific expression of the known insulin receptor isoforms generated by alternative splicing (insulin receptor types A and B), as assessed by polymerase chain reaction amplification with oligonucleotide primers flanking exon 11, was not correlated with the differences in the IC50 values and ratios for insulin and B29,B29'-suberoyl-insulin. Furthermore, IC50 values of both insulin and the B29 dimer were 3-fold lower in membranes from Rat1 cells overexpressing insulin receptor type A, compared with membranes with insulin receptor type B; the IC50 ratios were identical. No additional alternative splicing of insulin receptor mRNA was found by polymerase chain reaction amplification and digestion with HaeIII and AluI of seven overlapping domains of the receptor alpha subunit. These data suggest a heterogeneity of insulin receptors in rat tissues that is unrelated to alternative splicing of the insulin receptor gene.

Adipose Tissue↗
PubMed1992

Detection of a new hormone contact site within the insulin receptor ectodomain by the use of a novel photoreactive insulin.

We have used a preparation of soluble human insulin receptor ectodomain and a novel photoreactive, biotinylated derivative of insulin (4-azidosalicyloyl(B1-biocytinyl-B2-lysine)-insulin) to identify a new hormone contact site within the extracellular domain of the insulin receptor. The ectodomain was photoaffinity-labeled and digested to completion with trypsin, and the resulting tryptic fragment was purified by either HPLC or by streptavidin-affinity chromatography. The amino terminus of the fragment was identified as Gly390 within the alpha-subunit. These results suggest that residues that are carboxyl-terminal to the cysteine-rich domain, in addition to previously identified regions within the amino terminus of the alpha-subunit, contribute to the insulin binding site. The implications of these results for the de novo folding of the insulin receptor to constitute the hormone binding site are discussed.

Affinity Labels↗
PubMed1992

Design and synthesis of a novel biotinylated photoreactive insulin for receptor analysis.

B1-(4-Azido-salicyloyl)-[B1-biocytin,B2-lysine]insulin was synthesized by double Edman degradation of A1,B29-Msc2-insulin and stepwise acylation at the N-terminus of the B-chain. This derivative is homogeneous in RP-HPLC and has a biological in vitro activity of 20% and receptor binding of 15%, relative to insulin. Radioiodination and HPLC gave the B1-labelled 125I-derivative (I) as well as the 4 isomers with 125I-labelled tyrosine (A14, A19, B16, B26). UV-induced crosslinking of I with insulin receptors led to specific labelling of the alpha-subunit (Mr 130,000). The peptide bond LysB2-AspB3 is completely cleavable by trypsin (EC 3.4.21.4). I is thus a new tool for the analysis of the hormone-binding region by making possible the isolation of tryptic, biotinylated receptor fragments labelled by the dipeptide 125I-4-azidosalicyloyl-biocytinyl-Lys.

Affinity Labels↗
PubMed1992

Analysis of the human insulin receptor.

The insulin derivative 4-azidosalicyloyl-[B1-biocytin-B2-lysine]insulin was used to photo-affinity-label the highly purified insulin receptor from human placenta. As shown by SDS-polyacrylamide gel electrophoresis, the 5 monoiodo isomers, with iodine in positions B1, B16, B26, A14 or A19, gave different labelling patterns. After complete tryptic digestion of the covalent receptor complex with 125I-Asa-[BctB1,LysB2]insulin, a stable fragment of 18 kDa was isolated, which was further purified by HPLC. This tryptic fragment of the intact receptor corresponds, according to HPLC, Tricin-SDS-PAGE and 2D-electrophoresis, to the similarly labelled sequenced domain of the receptor ectodomain (Fabry, M. et al. (1992) J. Biol. Chem. 267, 8950-8956). We thus conclude that insulin is bound to identical contact sites of native receptor and truncated ectodomain.

Affinity Labels↗
PubMed1990

Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.

Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.

Amino Acid Sequence↗
PubMed1990

The detection of single cells forming antibodies to defined epitopes on insulin.

A solid-phase immunoenzymatic technique was modified to permit the detection and enumeration of antibody-secreting cells recognizing the amino or carboxy terminal of the insulin B chain. The procedure involves coating well surfaces with avidin and binding insulins specifically labelled with biotin at the B1 or B30 residue. On day 15 after immunization with human insulin, 20 outbred NMRI mice had generated cells secreting anti-insulin antibodies. The recognition of B1- or B30-related epitopes differed between individuals, suggesting that there was genetic determination of the epitopes expressed early in the immune response. The method can be used to find strains of mice with a preferential immune response to defined areas on the surface of small peptide molecules. Such strains could then be used to produce specific monoclonal antibodies.

Animals↗
PubMed1987

Survival rates and properties of sickle cell anemia red cells treated with nitrogen mustard.

A machine for the extracorporeal delivery of covalent anti-sickling agents has been described by Babb and coworkers and this therapeutic modality has been found feasible by others. We report here, further evaluation of nitrogen mustard (HN2), for possible use in the extracorporeal therapy of sickle cell anemia. Heparinized aliquots of whole blood from three patients with sickle cell disease were treated extra-corporeally with HN2 (0.65 mg/ml); the excess HN2 was neutralized with thiosulfate, and the blood was returned to the donor after labelling with 51Cr. HN2 produced an average 51Cr T1/2 which was 160% of control values. Fractionation of labelled red cells into different densities (ages) revealed that the main effect of HN2 was on the younger cell population which was characterized by a 51Cr T1/2 increase of 300% over the untreated blood. Further, "in vitro" studies were conducted to establish the effects on the SS red cells that could be expected from the treatment with HN2 at the concentrations used. Solubility determination (Csat) of deoxy hemoglobin S gels demonstrated that HN2 markedly inhibited the polymerization of HbS, was significantly more effective than potassium cyanate, and is among the most potent anti-sickling agents thus far reported. The viscosity of deoxygenated sickle red cells in a cone plate viscometer was markedly reduced by HN2, and hemodynamic studies using the microvasculature of an isolated rat mesoappendix demonstrated a reduction of peripheral resistance and an increase in flow rate when deoxygenated sickle cells pre-treated with HN2 were tested. At the highest concentration of HN2 (2 mg/ml), the peripheral resistance and the flow rates of deoxygenated cells attained levels found with oxygenated sickle blood. O2 affinity was partially corrected in the HN2 treated HbSS red cells. -SH reactivity of the red cell membrane was not affected by HN2. Among the added advantages of HN2 for extra-corporeal use is its rapid reaction rate, the lack of significant change of the O2 equilibrium at the concentration tested and the fact that the unreacted compound can be readily detected and neutralized. For these reasons HN2 is well suited for extra-corporeal treatment of sickle cell anemia for those patients with a severe form of the disease.

Anemia, Sickle Cell↗
PubMed1985

Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases.

We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.

Amino Acid Sequence↗
PubMedDate not supplied

Inhibition of HIV protease by monoclonal antibodies.

The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.

Animals↗
PubMedDate not supplied

Synthesis and characterization of photoreactive insulin-like growth factor 1 derivatives for receptor analysis.

Recombinant human insulin-like growth factor 1 (hIGF-1) was reacted with 4-azidosalicylic acid (Asa) to synthesize photoreactive IGF-1 derivatives. Depending on the time of reaction and the Asa/IGF-1 ratio, a mixture of mono-, bis-, and trisacylated derivatives and one tetraacylated derivative was produced, which was separated by reversed-phase HPLC. HPLC, PAGE, and MALDI mass spectrometry were used to determine the degree of the acylation and location of the photolabel insertion. After iodination of the three monoacylated photoreactive IGF-1 derivatives, the specific labeling of the receptor could be proved. Together with a comparative investigation in which B29-Asa-insulin was used, the results suggest corresponding contact areas for IGF-1 and insulin with the insulin receptor ectodomain.

3T3 Cells↗