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Biomedical subjects

B H Frank

Publications and source records attributed to B H Frank.

At least 91 records · Page 5Linked to original sources
PubMed1984

The degradation of monoiodotyrosyl insulin isomers by insulin protease.

The four single-site monoiodotyrosyl insulin isomers were synthesized by lactoperoxidase-catalyzed iodination of porcine insulin and were separated from one another by high performance liquid chromatography. The susceptibility of the four isomers (A14-, A19-, B16-, and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloroacetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatograpy. Using trichloroacetic acid precipitation, isomer susceptibility, determined from the initial rate of hydrolysis, was highest with the A14 isomer, lowest with the A19 isomer, and intermediate and roughly equal with the two B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Michaelis Menten constant (Km) of insulin protease was higher for the B16 isomer (55 nM) than for the other three isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). In addition, the values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. However, during incubation, the order of isomer susceptibility to insulin protease changed to B26 greater than A14 greater than A19 greater than B16. This change in apparent isomer susceptibility was prevented by including in the incubation mixture a rat renal peptidase, which did not degrade the intact isomers, suggesting that insulin protease converted the isomers to trichloroacetic acid-soluble products via trichloroacetic acid-precipitable intermediates. Using the immunoprecipitation assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was degraded more rapidly. Each isomer was converted more rapidly to nonimmunoprecipitable products than to trichloroacetic acid-soluble products, implying that insulin protease converted the isomers to trichloroacetic acid-precipitable, nonimmunoprecipitable intermediates, which it then converted to trichloroacetic acid-soluble form. Using Sephadex G-50 chromatography (SGC) assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was hydrolyzed more rapidly. With the exception of the A19 isomer, isomer hydrolysis appeared faster with SGC assay than with either of the other two assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Precipitation↗
PubMed1984

Human proinsulin-specific antigenic determinants identified by monoclonal antibodies.

Antigenic determinants recognized by human proinsulin (HPI)-specific monoclonal antibodies (Mabs) and Mabs crossreacting with free human C-peptide (HCP) were mapped by using various forms of purified, partially converted HPI intermediates. Two HPI-specific mouse Mabs (GS-4G9 and GS-9A8) reacted with the same antigenic determinant, GS, which was localized to the site of linkage of the B-chain to the C-peptide (Arg-Arg) at positions 31-32. These antibodies bind with equal efficiency to C65-A1 split proinsulin and to intact HPI. The binding of C32-C33 split proinsulin is markedly reduced. A rat Mab (GN-VIIB6), which crossreacts with free HCP in addition to HPI, reacted similarly with various HPI intermediates as it had with the corresponding synthetic HCP fragments, as previously reported (see ref. 9). This determinant (GN) is a three-dimensional structure composed of residues located in two separate regions in the C-peptide segment (positions 40-45 and 57-63). Reduced, carboxymethylated HPI retains the GN-determinant, whereas all insulin-like immunoreactivity identified with a conventional guinea pig insulin antiserum is completely lost. The binding of the two GS Mabs to the denatured HPI was reduced by 40-50% compared with intact HPI. It is concluded that the strong GN-determinant can readily form in the C-peptide segment of HPI, independently of the presence of ordered structure in the insulin moiety. A predicted beta-turn at position 47-50 may play an important role in bringing N- and C-terminal regions of the C-peptide segment into close proximity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1984

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes.

The receptor binding characteristics and biologic protency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15 degrees C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 +/- 0.8 X 10(-7) M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 +/- 0.3 X 10(-9) M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37 degrees C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 +/- 0.4 X 10(-8) M, whereas the same response was observed at an insulin concentration of 5.2 +/- 1 X 10(-11) M. Proinsulin also demonstrated an antilipolytic potency that was less than 1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1984

In vitro characterization of biosynthetic human proinsulin.

The binding and biologic properties of human proinsulin produced by recombinant DNA technology have been determined. The biosynthetic human proinsulin was iodinated using lactoperoxidase and subsequently purified by HPLC to yield the [(125I)TyrA14]-proinsulin isomer. Using isolated rat adipocytes, biosynthetic human proinsulin was shown to have approximately 11% of the binding potency of native insulin. At 16 degrees C and 37 degrees C, the ED50 values of biosynthetic human proinsulin were 3.7 nM and 15 nM, respectively, which was significantly different from the insulin values of 0.4 nM and 1.7 nM, respectively. Kinetic analysis suggested that the decreased affinity of biosynthetic human proinsulin was due primarily to a decreased association rate rather than an increased dissociation rate. Similar to insulin, biosynthetic human proinsulin exhibited a decreased half-time of dissociation in the presence of insulin (16.7 nM) or proinsulin (111 nM); however, this negative cooperative effect was lost in the presence of high concentrations of proinsulin (11 microM). Biologic potency, assessed by measuring glucose transport in rat adipocytes, showed that biosynthetic human proinsulin had 10% of the biologic activity of insulin, suggesting close coupling between binding to receptors and membrane generated cellular response. By extracting cell surface bound proinsulin with acidic buffer, the amount of 125I-proinsulin that internalized following binding to surface receptors was measured. At equilibrium, 55% of the cell-associated radioactivity was internalized at 37 degrees C. When chloroquine-treated (200 microM) cells were incubated with 125I-proinsulin at 37 degrees C, a 1.5-fold increase in the amount of intracellular proinsulin was observed at 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

High-performance liquid chromatographic preparation of single-site carrier-free pancreatic polypeptide hormone radiotracers.

The preferred radiotracer for use in radioimmuno- and receptor assays is one which is as similar as possible to the native hormone in both physicochemical and biological properties. Until recently, rapid detailed evaluation of iodination methodology as well as separation of site-specific tracers and their evaluation relative to the native protein has not been possible. Using reversed-phase high-performance liquid chromatographic methodology (C8 or C18 columns) we have evaluated a variety of iodination techniques and conditions. Procedures were found that allow the isolation of site-specific radiolabelled protein hormones in a rapid, reproducible and quantitative manner. The radiotracers are of the highest possible specific activity, have very low levels of damage and are more stable than tracers prepared by conventional techniques. This methodology has been applied to preparing tracers of beef, pork and human insulins, proinsulins and C-peptides as well as analogues of these proteins. In addition, the methodology has been applied to somatostatin, glucagon, pancreatic polypeptide, vasoactive intestinal peptide and luteinizing hormone-releasing hormone. The use of these tracers yielded increased sensitivity and reproducibility in a number of radioimmunoassays (e.g. C-peptide and glucagon), more reproducible results in radioreceptor assays (e.g. insulin receptors) and more defined studies on drug absorption and degradation. Finally, scale-up of our isolation procedures has yielded the 127I-analogue for homologous binding and displacement studies.

Animals↗
PubMed1983

Characteristics of binding of a low affinity, noncooperative insulin [( LeuB25]insulin) to IM-9 lymphocytes.

[Leu-B25]insulin is a low affinity insulin analog which does not increase the rate of dissociation of 125I-insulin from insulin receptors (i.e. does not display negative cooperativity). We have studied the characteristics of binding of this analog to IM-9 cultured lymphocytes, in order to determine the contribution of negative cooperativity to the curvilinear nature of Scatchard plots typical of insulin binding data. The affinity of [LeuB25]insulin for receptors was approximately 1% that of insulin, as determined by its ability to inhibit 125I-insulin binding. Monoiodinated preparations of insulin and of [LeuB25]insulin were produced, labeled in the tyrosine at position 14 of the A chain. These 125I-TyrA14-labeled species were used in all studies. Both native insulin and a serum containing antiinsulin receptor antibodies were equally potent at inhibiting binding of 125I-[LeuB25]insulin and 125I-native insulin, suggesting that they bind to the same population of receptors. Native insulin (100 ng/ml) increased the rate of dissociation of both 125I-insulin and 125I-[LeuB25]insulin. However, [LeuB25]insulin (2.5 micrograms/ml) did not increase the rates of dissociation of either 125I-insulin or 125I-[LeuB25]insulin (i.e. it did not display negative cooperativity). Competition curves and Scatchard plots were constructed using 125I-[LeuB25]insulin and unlabeled analog. Half-maximal inhibition of 125I-[LeuB25]insulin binding was seen at a [LeuB25]insulin concentration of approximately 500 ng/ml. More importantly, the Scatchard plot of these binding data was markedly curvilinear, as is typical of insulin binding data. In summary, a non-cooperative insulin analog, [LeuB25]insulin, yielded curvilinear Scatchard plots, indicating that negative cooperativity is not the full explanation of the curvilinear nature of Scatchard plots of insulin binding data. Therefore, an alternative explanation, such as the existence of heterogeneous receptor states, with differing affinities for the hormone, must contribute to the nonlinearity of these plots.

Cell Line↗
PubMed1975

Ionic strength effects on the stability and conformation of Penicillium chrysogenum mycophage double stranded RNA.

The effect of [Na+] on the stability and conformation of penicillium chrysogenum mycophage dsRNA (PCMdsRNA) was investigated using CD and UV optical techniques. Thermal melting profiles reveal prominent fine structure attributed to at least four regions of structural dissimilarity. A constant increased thermal stability of the dsRNA compared to DNA of the same base composition was observed over a concentration range of 1.5 times 10- minus 4 M to 4.5 times 10- minus 2 M Na+. At low ionic strengths ([Na+] less than 10- minus 3 M) Tm becomes independent of further decrease in [Na+] unless the dsRNA is exposed to high concentrations of EDTA, suggesting the involvement to tightly bound divalent cation. At relatively high ionic strengths ([Na+] greater than 0.1 M) a postulated A leads to A' ... conformation change occurs.

Circular Dichroism↗
PubMed1972

Differences in the nature of the interaction of insulin and proinsulin with zinc.

1. The reversible interaction of zinc with pig insulin and proinsulin has been studied at pH7 by equilibrium dialysis (ultrafiltration) and by sedimentation equilibrium and velocity measurements in the ultracentrifuge. Binding values calculated from equilibria, where the ratio of free to bound zinc was varied in the range 0.01:1-10:1, indicated that proinsulin and insulin each contained two main orders of zinc binding with very different affinities for the metal. 2. In equilibria containing low concentrations of free zinc (free: bound ratios of 0.01-0.1:1) both insulin and proinsulin aggregated to form soluble hexamers containing firmly bound zinc (up to 0.284g-atom/monomer) with an apparent intrinsic association constant of 1.9x10(6)m(-1). 3. Higher concentrations of zinc (free: bound ratios of 0.1-10.0:1) resulted in a progressive difference in the zinc binding, aggregation and solubility properties of the metal complexes of insulin and proinsulin. At the highest concentration of free zinc, proinsulin bound a total of more than 5.0g-atom/monomer and aggregated to form a mixture of soluble polymers (mainly 5.1S). In contrast, insulin bound a total of only 1.0g-atom/monomer and was almost completely precipitated from solution. 4. These results would indicate that the presence of the peptide segment connecting the insulin moiety in proinsulin does not prevent the firm binding of zinc to the insulin moiety and the formation of hexamers of zinc-proinsulin. At the same time although the connecting peptide contains additional sites of lower affinity for zinc, which should facilitate inter- and intra-molecular cross-linking, the general conformation of the zinc-proinsulin hexamer must preclude the formation of very large and close-packed aggregates that are insoluble in solutions at equilibrium.

Animals↗