Biomedical subjects

J Markussen

Publications and source records attributed to J Markussen.

At least 55 records · Page 3Linked to original sources

PubMed1976

Separation of the two double-chain bovine intermediates of the proinsulin-insulin conversion I. Chemical, immunochemical, circular dichroism, and biological characterization.

The intermediates of the proinsulin-insulin conversion were separated by cation exchange. The circular dichroism spectra of the intermediates showed less alpha-helix than insulin and proinsulin. It is suggested that the C-peptide interacts with the section of alpha-helix contained between residues 2 and 8 in the A-chain of the insulin moieties and unwinds the alpha-helix. The in vivo activities of the intermediates were found to be in the order of 50% relative to insulin. In the fat cell assay, the A-chain-substituted form is weaker (9%) than the B-chain-substituted form (19%). The C-peptide segments of the two forms reacted with C-peptide-specific antibodies as fully as the free C-peptide, on a molar basis. In contrast, the insulin segments were hindered from reacting with insulin-specific antibodies as fully as the insulin.

Amino Acids↗

PubMed1976

Production of antisera to synthetic benzyloxycarbonyl-C-peptide of human proinsulin.

Antisera to the C-peptide of human proinsulin were obtained by immunizing guinea pigs with synthetic benzyloxycarbonyl-C-peptide conjugated to human albumin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In three series of 10, the animals were injected with C-peptide conjugated to albumin in the molar ratio of 23 : 1, 15 : 1, and 4 : 1, respectively. Antibodies to human C-peptide were present in all the surviving 25 animals. Fifteen of the antisera were suitable for measuring C-peptide concentrations lower than 0.10 pmol/ml. The antisera demonstrated an increasing immunogenicity with increasing molar ratio of C-peptide to albumin in the conjugate. In the fourth series, ten guinea pigs immunized with benzyloxycarbonyl-C-peptide ionically bound to QAE-Sephadex A-25 did not produce detectable antibodies to C-peptide. A qualitative evaluation of the radioimmunoassay by use of the antiserum with the highest titer and sensitivity, "M 1230", revealed a mean intra-assay and inter-assay coefficient of variance of 3.2 and 9.6%, respectively.

Animals↗

PubMed1975

Conformational analysis of circular dichroism spectra of insulin, proinsulin and c-peptides by non-linear regression.

A method of resolving CD spectra in alpha-helix, beta-structure and random coil conformations is described. The residue ellipticites for alpha-helix and beta-structure given by Greenfield & Fasman or by Chen,, Yang & Martinez are used together with CD spectra from at least two similar peptides to determine, by an iterative least-squares method, the number of amino acids in the three reference conformations as well as a set of residue ellipticities characteristic of the random coils of the family of peptides in question, but not necessarily of other peptides. The fits between computed and experimental spectra improve significantly and systematic deviations disappear by allowing the random coil coefficients to vary from one family of proteins to another, a liberty justified by the different types of random coils that have been encountered. The method of analysis showed that 5 M urea did not change the conformations of C-peptides of proinsulin from ox, pig and duck, all being mainly in the random coil conformation and all having 3-4 amino acids in beta-structure. Bovine insulin and proinsulin showed a transfer of amino acids from alpha-helix to beta-structure with increasing concentrations of urea, the latter at a higher concentration, indicating a stabilizing effect of the connecting peptide. The numbers of amino acids found in the alpha-helical conformation in insulin and proinsulin were equal and in agreement with the X-ray crystallographic data for insulin when the Greenfield & Fasman coefficients for alpha-helix and beta-structure were employed, whereas the Chen, Yang & Martinez coefficients yielded too few amino acids in alpha-helix in proinsulin. Both sets of coefficients estimate more beta-structure in proinsulin than in insulin.

Amino Acids↗

PubMed1975

Studies on polypeptides, VI. Synthesis, circular dichroism and immunological studies of tyrosyl C-peptide of human proinsulin.

The synthesis of tyrosyl human C-peptide, a sequence of 32 amino acids, by the fragment condensation of the N-terminal octapeptide and C-terminal tetracosapeptide is described. The t-butyl protecting groups were removed by trifluoroacetic acid to obtain N-benzyloxycarbonyl-tyrosyl C-peptide. The hydrogenolytic debenzyl-oxycarbonylation of this derivative proceeded to an extent of only 80-90%, and tyrosyl C-peptide was purified by preparative electrophoresis. This purified tyrosyl C-peptide led to an improved sensitivity of the radioimmunoassay. The synthetic tyrosyl C-peptide in an immunoassay using anti human b-component serum reacted slightly differently from the synthetic human C-peptide. After labelling tyrosyl C-peptide with 125I and then purifying the radioactive product, we observed that 80% of the radioactivity could be bound when reacted with an excess of the serum. The circular dichroism spectrum of tyrosyl C-peptide is very similar to that of synthetic human C-peptide. An analysis of the spectrum indicates that 3-7 amino acids are in the beta-structure and the rest in random coil conformation.

Amino Acid Sequence↗

PubMed1975

Studies on polypeptides. V. Improved synthesis of human proinsulin C-peptide and its benzyloxycarbonyl derivative. Circular dichroism and immunological studies of human C-peptide.

An improved synthesis of human C-peptide is described. Five fragments: 33-39, 40-46, 47-49, 50-54 and 55-63 were used in the total synthesis. In the fully protected C-peptide the N-terminal alpha-amino function was blocked by a benzyloxycarbonyl group and the carboxyl and serine hydroxyl functions were blocked by t-butyl protection. The latter protecting groups were removed by trifluoroacetic acid to obtain N-alpha-benzyloxycarbonyl human C-peptide which, on catalytic hydrogenation, yielded human C-peptide. The immunoreactivity of the prepared human C-peptide was tested and found to deviate slightly from the human C-peptide synthesized earlier by another route. When tested in the immunoassay, human pancreatic extracts containing natural C-peptide (or fragments thereof) showed dilution patterns identical to that of the new synthetic C-peptide but different from that of the previously synthesized batch of C-peptide. The possible explantation for the observed differences in the immunoreactivity is discussed.

Benzyl Compounds↗

PubMed1974