Familial hyperproinsulinemia. Two cohorts secreting indistinguishable type II intermediates of proinsulin conversion. (57/78)

Familial hyperproinsulinemia, a hereditary syndrome in which individuals secrete high amounts of 9,000-mol wt proinsulin-like material, has been identified in two unrelated cohorts. Separate analysis of the material from each of the two cohorts had suggested that the proinsulin-like peptide was a conversion intermediate in which the C-peptide remained attached to the insulin B-chain in one case, whereas it was a conversion intermediate in which the C-peptide remained attached to the insulin A-chain in the other. To reinvestigate this apparent discrepancy, we have now used chemical, biochemical, immunochemical, and physical techniques to compare in parallel the structures of the immunoaffinity chromatography-purified, proinsulin-like peptides isolated from the serum of members of both families. Our results show that affected individuals in both cohorts secrete two-chained intermediates of proinsulin conversion in which the COOH-terminus of the C-peptide is extended by the insulin A-chain and from which the insulin B-chain is released by oxidative sulfitolysis. Analysis of the conversion intermediates by reverse-phase high-performance liquid chromatography using two different buffer systems showed that the proinsulin-related peptides from both families elute at a single position very near that of the normal intermediate des-Arg31, Arg32-proinsulin. Further, treatment of these peptides with acetic anhydride prevented trypsin-catalyzed cleavage of the C-peptide from the insulin A-chain, a result demonstrating the presence of Lys64 and the absence of Arg65 in both abnormal forms. We conclude that individuals from both cohorts with familial hyperproinsulinemia secret very similar or identical intermediates of proinsulin conversion in which the C-peptide remains attached to the insulin A chain and in which Arg65 has been replaced by another amino acid residue.  (+info)

Nature of the interaction between Ricinus communis agglutinin and blood cells. (58/78)

Binding of Ricinus communis agglutinin (RCA 120) to carbohydrate receptors of human lymphocytes and erythrocytes is enthalpically driven. As in the case of simple saccharides, the delta S contribution is always unfavorable to the interaction. This result is different from that observed for other lectins and might indicate that hydrophobic interactions do not play a dominant role in binding of RCA 120 to cell surfaces.  (+info)

Quantitative investigation of rapid injector port derivatization of amphetamine using trifluoroacetic anhydride with packed and capillary column GC and GC/MS methods. (59/78)

Direct injector port derivatization of amphetamine through coinjection of sample with trifluoroacetic anhydride was quantitatively investigated and found to be a rapid and more sensitive alternative to longer solution-based derivatization procedures. Under optimum conditions determined in this work, limits of detection were 1 ng using gas chromatography with flame ionization detection and 1 pg using single ion monitoring of ion 140.0 amu (M-91+) in gas chromatography/mass spectrometry methods. The previously reported derivatization period of over 12 hr was greatly reduced using instantaneous derivatization with a 100- to 1000-fold increase in limits of detection. Effects of inlet temperature, mass of trifluoroacetic anhydride, and column pre-treatment on chromatographic performance are reported. Application to analysis of serum from amphetamine-dosed rats was used for comparison to accepted solution-based derivatization.  (+info)

Differential susceptibility of mono- and di-O-alkyl ether phosphoglycerides to acetolysis. (60/78)

The effectiveness of acetolysis as a tool in structural characterization of mono- and di-O-alkyl phosphoglycerides was investigated. Surprisingly, it was found that the di-O-alkyl phosphoglycerides were resistant to attack during acetolysis, whereas the mono-ether types, with a free hydroxyl function or an ester on carbon-2, were easily attacked at the glycerol-phosphate bond. On the other hand, Vitride reduction occurred readily with the mono-ether or di-ether phosphoglycerides. The implications of these findings as they relate to identification of ether phospholipids in tissues are discussed.  (+info)

Study of the structure of troponin-I by measuring the relative reactivities of lysines with acetic anhydride. (61/78)

A competitive labeling method that measures the relative reactivity of lysines was used to study the structure of troponin-I. Troponin-I was acetylated free and complexed with troponin-C and troponin-T in the native state with [3H]acetic anhydride. The [3H]troponin-I was combined with [14C]troponin-I that had been acetylated in 6 M guanidine HCl and completely chemically labeled. Peptides containing labeled lysines were isolated following digestion with trypsin and Staphylococcus aureus protease and identified in the published sequence. The 3H/14C ratio of these peptides was used as a measure of the relative reactivity of the lysines. Troponin-I contains 24 lysines; we have identified 23 of these in 16 peptides. When troponin-I is labeled in a native complex, the lysines in the region from residues 40 to 98 are influenced: five become relatively less reactive (40, 65, 70, 78, and 90) and three become relatively more reactive (84, 87), and 98). All of these changes except Lys 70 can be seen when troponin-I binds to troponin-T. Lys 70 is reduced in reactivity when it binds to troponin-C. The lysines that appear to be important in binding of troponin-I to troponin-T are influenced by the binding of Ca2+ to troponin-C in the native troponin complex (in the presence of 2 mM MgCl2), suggesting for the first time that the troponin-IT interaction is affected by Ca2+.  (+info)

Changes in actin lysine reactivities during polymerization detected using a competitive labeling method. (62/78)

We have studied the structure of actin by measuring the relative reactivities of lysines with acetic anhydride using a competitive labeling procedure comparing monomeric globular actin. monomeric actin in the presence of salt, and filamentous actin polymerized in 100 mM NaCl and 100 mM NaCl, 2 mM MgCl2. We have identified 12 of the 19 lysines: 18, 50, 61, 68, 113, 191, 237, 290, 315, 325, 327, and 358. In all conditions, Lys (325, 327) is the most reactive. In globular actin, Lys 18, 191, 290, 314. and 358 are less than 20% as reactive as Lys (325, 327); the remaining have intermediate reactivities. On polymerization in the presence of NaCl and Mg2+, lysines 50, 61, 68, 113, and 290 become less reactive relative to Lys (325, 327). The changes in Lys 50, 61, and 113 are due largely to the polymerization event whereas those in Lys 68 and 290 appear to be an effect of Mg2+. Lys 18, 191, and 358 increase in relative reactivity when cation is added to the monomer and then become less reactive in the polymer, showing no large overall change in reactivity relative to the monomer in the absence of salt. Lysines that are reduced in reactivity upon polymerization indicate possible contact regions between actin monomers in the filament in the NH2-terminal third of the protein.  (+info)

Transient conformational states in proteins followed by differential labeling. (63/78)

Refolding of previously denatured and reduced elastase has been followed by titration of chemical reactivities of amino acid side chains to study the topography of the protein in the native state, and the microenvironment variations of protein side chains during the structural transition. Groups accessible to chemical reagents in the denatured form and buried in the "native" form were used as a local conformational probe. Times of labeling, depending on the reagent used, ranged from 100 to 800 ms. The reaction was stopped by isotopic dilution with an excess of unlabeled reagent under denaturing conditions to obtain a chemically homogeneous but heterogeneously labeled material. Peptide fractionation after degradation of the labeled proteins allowed the determination of the amount of radioactive label incorporated by the individual side chains during the refolding. Refolding rates, determined by physicochemical, enzymatic or immunochemical criteria, were compared with the conformational states of protein areas and evaluated by the variation of chemical reactivity at various denaturant concentrations. The importance of the last folding stages is emphasized by the results obtained which indicate that early during the refolding, two domain substructures (H-40 to H-71 and M-180 to H-200)( are stabilized, while the protein remains inactive at the time ranges of the labeling reactions.  (+info)

Chemical properties of the functional groups of insulin. (64/78)

The method of competitive binding [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] with 1-fluoro-2,4-dinitrobenzene as the labelling reagent [Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175] was used to determine the chemical properties, namely pK and reactivity, of the amino groups, the histidine residues and the tyrosine residues of the dimeric form of pig zinc-free insulin at 20.0 degrees C. The N-terminal glycine residue of the A-chain has a pK of 7.7 and a slightly higher than normal reactivity. The N-terminal phenylalanine residue of the B-chain has a pK of 6.9 and is approximately an order of magnitude more reactive than a corresponding amino group with the same pK value. The lysine epsilon-amino group has an unusually low pK of 7.0 but has approximately the expected reactivity of such a group. In the case of the two histidine and four tyrosine residues only the average properties of each class were determined. The histidine residues have a pK value of approx. 6.6, but, however, their reactivity is at least an order of magnitude greater than that of a free imidazole group. The tyrosine residues have a pK value of approx. 10, but their average reactivities are substantially less than for a free phenolic group. At alkaline pH values above 8 the reactivity of all the functional groups show sharp discontinuities, indicating that insulin is undergoing a structural change that alters the properties of these groups.  (+info)