Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis.

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near, apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38,000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase: the order of peptide bond formation and timing of the epimerisation reaction.

delta-L-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase catalyses the formation of the common precursor tripeptide of both the penicillin and cephalosporin antibiotics from the L-enantiomers of its constituent amino acids. Replacement of cysteine with L-O-methylserine in preparative-scale incubations led to the isolation of both L-O-methylserinyl-L-valine and L-O-methylserinyl-D-valine dipeptides. The dipeptides were characterized with the aid of authentic synthetic standards by both 1H NMR and electrospray ionization MS. A revised mechanism for ACV biosynthesis involving formation of the cysteinyl-valine peptide bond before the epimerisation of valine and subsequent condensation with the delta-carboxyl of L-alpha-aminoadipate is therefore proposed.

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins.

Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.

Insulin deficiency in non-insulin-dependent diabetes.

A highly specific two-site immunoradiometric assay for insulin was used to measure the plasma insulin response to 75 g glucose administered orally to 49 patients with non-insulin-dependent diabetes (NIDDM). The plasma insulin concentration 30 min after glucose ingestion was lower in the diabetic patients than in matched controls for both non-obese (11-83 pmol/l vs 136-297 pmol/l, p less than 0.01) and obese subjects (23-119 pmol/l vs 137-378 pmol/l, p less than 0.01). By means of another two-site immunoradiometric assay, the basal intact proinsulin level was found to be higher in the NIDDM patients than in the controls for both non-obese (7.1 [SEM 1.2] pmol/l vs 2.4 [0.4] pmol/l, p less than 0.01) and obese subjects (14.4 [2.2] pmol/l vs 5.9 [1.9] pmol/l, p less than 0.01). The basal level of 32-33 split proinsulin was also raised in NIDDM. Previous failure to show clear separation between normal and NIDDM insulin responses was probably due to the high concentrations of proinsulin-like molecules in the plasma of NIDDM patients. These substances cross-react as insulin in most, if not all, insulin radioimmunoassays but have very little biological insulin-like activity. It is therefore now possible and necessary to designate most NIDDM patients as insulin deficient.