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J Markussen

Publications and source records attributed to J Markussen.

At least 19 recordsLinked to original sources
PubMed1992

Role of B13 Glu in insulin assembly. The hexamer structure of recombinant mutant (B13 Glu-->Gln) insulin.

The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.

Animals↗
PubMed1992

Application of the euglycaemic clamp technique to bioassay of insulin analogues.

The euglycaemic clamp method may offer a precise and clinically valid approach to assess the in vivo potency of new insulin analogues or derivatives relative to a human insulin standard. The proposed protocol was designed to overcome problems due to differences in pharmacokinetics between the test and standard preparations. An analogue of human insulin, GlyA21+ArgB27+ThrB30-NH2, which is absorbed very slowly after subcutaneous injection, and human insulin were compared in intravenous clamp experiments in pigs. Both insulins were infused for 4 h to achieve steady state glucose metabolism. The infusion rate ranged from 2.5-8 pmol min-1 kg-1. Parallel dose response curves were obtained with the mean glucose infusion rate from 180-240 min as the response and the logarithm of the insulin infusion rate as the dose. Standard bioassay analysis showed that the molar potency of the analogue relative to human insulin was 95.2% with a 95% confidence interval of 82.3-111.2%. To assess the clinical validity of the method a similar euglycaemic clamp study was carried out in human volunteers. The insulin infusion rates were 3 and 6 pmol min-1 kg-1, and the mean glucose infusion rate over the final 180-240 min period of the clamp was used as response. The statistical analysis showed, as in the pig clamp bioassay, no significant deviations from steady state or from the assumption of parallelism. The resulting molar potency of the analogue relative to human insulin was 85.5% with a 95% confidence interval of 49.5-128.4%. This was in agreement with the result of the pig clamp bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Insulin analogues with improved absorption characteristics.

The insulin preparations available today are not ideal for therapy as s.c. injection does not provide a physiological insulin profile. With the aim to improve the absorption properties recombinant DNA technology has been utilized to design novel insulin molecules with changed physico-chemical characteristics and hence altered subcutaneous absorption kinetics. Soluble, long-acting human insulin analogues in which the isoelectric point has been increased from 5.4 to approx. 7 are absorbed very slowly, providing a more constant basal insulin delivery with lower day-to-day variation than present protracted preparations. In addition they have better storage stability. Rapid-acting human insulin analogues with largely reduced self-association are absorbed substantially faster from subcutaneous tissue than current regular insulin and thus are better suited for bolus injection. The absorption kinetics of these analogues have been able to explain the mechanism behind the dose effect on insulin absorption rate.

Absorption↗
PubMed1991

Immobilized insulin for high capacity affinity chromatography of insulin receptors.

Insulin receptors can be purified by affinity chromatography on immobilized insulin, but published methods all suffer from a rather low capacity of the affinity columns. By using insulin that has been protected in positions A1 and B29, we have been able to couple the insulin selectively through the B1 amino group to divinyl sulfone-activated agarose. The N terminus of the B-chain is the most innocuous site as far as receptor-insulin interaction is concerned, and this strategy allowed us to make affinity columns with capacities of several milligrams of receptor/ml of resin. The receptor used was the soluble ectodomain of the human insulin receptor, produced in transfected baby hamster kidney cells. The column preparation and the elution conditions are described in detail, as the efficacy of the purification depends strongly on both. The purity of the eluted receptors was so high that quantitative amino acid analysis fitted with theory. The molar absorption coefficient at 278.5 nm was 296,000 M-1 cm-1. Finally, it could be unequivocally established that the soluble receptor binds two molecules of insulin with equal affinity.

Amino Acids↗
PubMed1991

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.

Amino Acid Sequence↗
PubMed1991

Reaction mechanism of trypsin-catalysed semisynthesis of human insulin studied by fast atom bombardment mass spectrometry.

The production of semisynthetic human insulin for therapeutic purposes is of considerable importance. During trypsin-catalysed transformation of pig insulin into an ester of insulin of human sequence, the alanyl residue at position B30 is removed and replaced with an esterified residue of threonine. We have carried out this transformation in a medium enriched in 18OH2 and studied the product by MS. In contrast to a previous report, we find that incorporation of label into the B29 - B30 peptide bond occurs during the transformation with threonine methyl ester in aqueous N,N-dimethylacetamide. Quantitative data are presented and the implications of these findings are discussed.

Animals↗
PubMed1991

In vitro and in vivo potency of insulin analogues designed for clinical use.

Analogues of human insulin designed to have improved absorption properties after subcutaneous injection have been prepared by recombinant DNA technology. Five rapidly absorbed analogues, being predominantly in mono- or di-meric states in the pharmaceutical preparation, and a hexameric analogue with very low solubility at neutral pH and slow absorption, were studied. Receptor binding assays with HEP-G2 cells showed overall agreement with mouse free adipocyte assays. Two analogues, B28Asp and A21Gly + B27Arg + B30Thr-NH2, had nearly the same molar in vitro potency as human insulin. Another two showed increased adipocyte potency and receptor binding, B10Asp 194% and 333% and A8His + B4His + B10Glu + B27His 575% and 511%, while B9Asp + B27Glu showed 29% and 18% and the B25Asp analogue only 0.12% and 0.05% potency. Bioassays in mice or rabbits of the analogues except B25Asp showed that they had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation in in vivo potency reflects the differences in receptor binding affinity. Relative to human insulin a low concentration is sufficient for a high affinity analogue to produce a given receptor complex formation and metabolic response. In conclusion, human insulin and analogues with markedly different in vitro potencies were equipotent in terms of hypoglycaemic effect. This is in agreement with the concept that elimination of insulin from blood and its subsequent degradation is mediated by insulin receptors.

Adipose Tissue↗
PubMed1989

NovoSol Basal: pharmacokinetics of a novel soluble long acting insulin analogue.

OBJECTIVE: To determine the courses of absorption and the interindividual and intraindividual variations in absorption of iodine-125 labelled Ultratard HM and NovoSol Basal injected subcutaneously. DESIGN: Open randomised crossover study. Each patient was tested during two study periods of five days each, during which he or she received a subcutaneous injection of either 125I-NovoSol Basal or 125I-Ultratard HM on four consecutive days. The aim was to detect a reduction in intraindividual standard deviation by a factor of two with a probability 0.95, taking 0.05 as the level of significance. This required 24 degrees of freedom and led to the choice of four courses in each of eight patients. SETTING: Referrals to the diabetes research centre in Hvidøre, Copenhagen. PATIENTS: Eight insulin dependent (type I) diabetics with low or undetectable C peptide concentrations who were receiving a multiple insulin injection regimen. One patient withdrew immediately after recruitment. INTERVENTIONS: After an overnight fast patients received 96 nmol (16 IU insulin) of either 125I-NovoSol Basal or 125I-Ultratard HM injected subcutaneously into the thigh. To ensure that the insulin entered the subcutaneous fat at the same depth, ultrasonography was performed on each patient before the first injection. A different injection site on the thigh was used each day for four days in order to facilitate monitoring of the disappearance of four different depots in each patient. MAIN OUTCOME MEASURE: Residual activity at the injection site was measured roughly every two hours throughout the day. No radioactivity measurements were performed overnight (10 pm till 8 am). The residual radioactivity after the injection on the first day (upper right thigh) was recorded for five days, that after the injection on the second day (upper left thigh) for four days, after the injection on the third day (lower right thigh) for three days, and after the last injection (lower left thigh) for two days. RESULTS: NovoSol Basal was absorbed according to first order kinetics with a mean t50% of 35.3 (SEM 1.4) hours; Ultratard HM was absorbed after a lag phase and the corresponding t50% was 25.5 (2.5) hours. The intraindividual variations in t50% were significantly smaller with NovoSol Basal than with Ultratard HM (18.4% v 44.5%; p less than 0.001). Interindividual variations, however, were not significantly different (25.2% v 36.9%; p = 0.38). The total variation in t50% was substantially smaller with NovoSol Basal than with Ultratard HM (20.3% v 42.8%). CONCLUSIONS: NovoSol Basal seems to be an appreciable advance over Ultratard HM as a soluble insulin preparation for obtaining reproducible 24 hour insulin concentrations in the blood

Absorption↗
PubMed1988

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30.

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

Absorption↗
PubMed1987

Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain.

Hydrophilic insulins, more positively charged than human insulin at neutral pH, have been prepared by substitution with basic amino acids at the termini of the B-chain and by blocking the C-terminal carboxyl group of the B-chain. The isoelectric pH of the insulin is thereby moved from 5.4 towards physiological levels. Slightly acid solutions of derivatives, in which charge has been added in the C-terminus of the B-chain, have a prolonged action in vivo, in particular if the carboxyl group is blocked. It is found that the prolonged-acting hydrophilic insulins crystallize instantly when the pH is adjusted to 7. The prolonged action is ascribed to this readiness to crystallization combined with a low solubility, which may be further decreased by increased concentration of zinc ions. Hydrophobic insulins have a prolonged action independent of the site of substitution even if the derivative is soluble at physiological pH. Some derivatives were prepared from porcine insulin by tryptic transpeptidation. N-terminal B-chain substituted insulins were prepared by alkylation of a biosynthetic single-chain insulin precursor, followed by tryptic transpeptidation rendering the double chain insulin derivative. The observed blood glucose lowering in the rabbits implies that neither N- nor C-terminal B-chain substitution results in substantial deterioration of biological potency. An index for the degree of protraction based on the blood glucose data is used to compare the insulins.

Animals↗
PubMed1987

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30.

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Isolation and amino acid sequence of insulins and C-peptides of European bison (Bison bonasus) and fox (Alopex lagopus).

Insulins and C-peptides were extracted and purified from bison and fox pancreatic glands. The insulins were reduced and pyridylethylated, and the derived A- and B-chains separated by HPLC. Amino acid sequence determinations of the pyridylethylated A- and B-chains proved bisontine insulin to be identical to bovine insulin and fox insulin to be identical to dog and porcine insulin. Bisontine C-peptide proved to be identical to bovine C-peptide. The isolated fox C-peptide comprises 23 amino acid residues and probably represents a major tryptic fragment of a larger C-peptide. The fox C-peptide fragment is identical to the dog C-peptide (9-31) except for residue 3 (residue 11 in the dog C-peptide), which is aspartic acid as compared with glutamic acid in the dog C-peptide.

Amino Acid Sequence↗
PubMed1985

Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21).

Glucagon-(1-21) was prepared fully synthetically as well as by carboxypeptidase A digestion of natural porcine glucagon. Neither of the two preparations had glucagon agonistic effects with regard to receptor binding or adenylate cyclase activation in purified rat liver plasma membranes. Nor did these preparations contain lipolytic activity in isolated free fat cells. A preliminary batch of glucagon-(1-21) prepared by carboxypeptidase A digestion did, however, contain 1-2% glucagon bioactivity. This activity was separated from glucagon-(1-21) by high-performance liquid chromatography and quantitatively recovered in four minor hind peaks which eluted close to but not in a position identical to the elution position of native glucagon.

Adenylyl Cyclases↗
PubMed1985

Kinetics of trypsin catalysis in the industrial conversion of porcine insulin to human insulin.

The kinetics of coupling and transpeptidation reactions catalysed by trypsin were studied in media with low water contents to see whether the usual Michaelis-Menten kinetics applied under the unusual conditions used in peptide bond synthesis, to obtain information about the magnitude of rate constants and activation energies, and to gain insight into the mechanism of catalysis. Porcine insulin, des-(AlaB30)-porcine insulin, human insulin-ThrB30-OMe and human insulin-ThrB30(But)-OBut were used as substrates. Two threonine esters (Thr-OMe and Thr(But)-OBut) were used for transpeptidation and coupling. The reactions progressed according to first-order kinetics until about 35% conversion, and the experimental data were adequately explained by Michaelis-Menten kinetics. The rates of the coupling and transpeptidation reactions in media with low water contents were orders of magnitude below the rates of peptide bond hydrolysis by trypsin in water. It was not possible to approach saturation of the enzyme with substrate so determination of Km was impossible, but for each substrate a value larger than 0.1 M was estimated from the Lineweaver-Burk plot. The rate of release of alanine from porcine insulin depended on the type of threonine ester present; for example, Thr-OMe inhibited the reaction. Coupling occurred faster than transpeptidation. However, in the medium used, the activation energies for the two reactions were similar (about 50 kJ/mol), so the difference in reaction rates is probably due to different transmission coefficients in the activated transition states. Computer simulations enabled us to obtain quantitative descriptions of the reaction progress curves from fitted rate constants.

Alanine↗
PubMed1985

Comparative reduction/oxidation studies with single chain des-(B30) insulin and porcine proinsulin.

The single chain des-(B30) insulin molecule (SCI) has been reduced and reoxidized together with porcine proinsulin (PPI). Yields of correctly folded and reoxidized SCI and PPI were analyzed by HPLC. The concentrations of both proteins were 10(-3) M during reduction and 10(-5) M during oxidation. The pH during reoxidation was varied from 8.6 to 9.2 and the temperature from 4 to 37 degrees. Under all conditions tested, the recovery of SCI was substantially higher than that of PPI. The recoveries peaked after 24-72 h. It is suggested that the "miniproinsulin" SCI folds correctly up more efficiently than porcine proinsulin, resulting in higher yields of reoxidized SCI.

Chromatography, High Pressure Liquid↗
PubMed1985

Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation.

Single chain des-(B30) insulin (SCI) has been synthesized from porcine insulin by trypsin in a medium with a low content of water. Trypsin catalyzes an intramolecular transpeptidation reaction in which the glycineA1 residue substitutes the alanineB30 residue, rendering a LysB29 -GlyA1 peptide link between the A- and B-chains of insulin. The insulin derivative has been purified by column chromatography and appears to be homogeneous in HPLC and disc electrophoresis. The structure was proven to be B(1-29)-A(1-21) insulin by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degradation. The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form. SCI was devoid of effect in the blood sugar lowering assay in mice, the estimated potency being less than 0.1% of that of insulin.

Amino Acids↗
PubMed1983

Human monocomponent insulin. Chemistry and characteristics.

The primary structure of different insulins is reviewed and the properties, identification tests, purity, potency and immunogenicity of human insulin are summerized. Novo Research Institute has developed a method, simply using an enzymatic conversion reaction to substitute the B30 alanine of porcine insulin with threonine to manufacture human insulin. This process is basically an extension of the process currently used to manufacture the Novo purified insulins which are commercially available.

Amino Acids↗