Photoaffinity labelling of isopenicillin N synthetase.

The alpha-aminoadipoyl group of the natural substrate of isopenicillin N synthetase (IPNS), L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), has been replaced by a diazirinyl-containing group, which can be photoactivated. This has allowed investigation of the substrate binding site of IPNS by photoaffinity labelling. Laser flash photolysis of this analogue, [3H]DCV, in the presence of IPNS leads to the incorporation of radioactivity into the enzyme. Tryptic digestion of this labelled enzyme, followed by separation and sequencing of the resultant fragments, identified two labelled regions of the protein. These are the fragments Asp-40 to Arg-78 and Thr-237 to Gly-256.

Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis.

Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

Characterization of the subunits of beta-conglycinin.

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.

Inheritance and biochemical analysis of four electrophoretic variants of ß-conglycinin from soybean.

Three genes which code for variant ß-conglycinin subunits were identified. Alleles Cgy 1 (S) and Cgy 2 (S) were codominant with Cgy 1 and Cgy 2 and produced α' and α subunits, respectively, with reduced electrophoretic mobility. Allele Cgy 3 (D) increased the mobility of at least one polypeptide in the ß subunit family and exhibited incomplete dominance. Gene loci Cgy 2/Cgy 2 (S) and Cgy 3 (D) /cgy 3 (D) were linked, whereas Cgy 1/Cgy 1 (S) / cgy 1 segregated independently of the others. Techniques developed for purification of normal ß-conglycinin subunits were effective in purifying the altered subunits. Deglycosylated variant proteins from seeds containing the alleles Cgy 1 (S) , Cgy 2 (S) , or Cgy 3 (D) also has altered mobility relative to deglycosylated normal proteins. Therefore, the altered subunits contained changes in their amino acid sequences rather than in their carbohydrate moieties. This interpretation is consistent with the observed codominant or incompletely dominant mode of inheritance for these alleles and suggests that each contains an altered nucleotide sequence in the structural gene. A fourth variant, which exhibited doublet α' and a electrophoretic bands, was inherited in a recessive fashion. Deglycosylated subunit proteins from this variant were identical in electrophoretic mobility to those of the deglycosylated normal protein. This suggests that the doublet phenotype resulted from an alteration in the carbohydrate moiety of these subunits. The gene or genes which condition this variant presumably are required for normal post-translational modification of the subunit carbohydrates and as such may be useful for investigating these events.

The molecular basis of the selectivity of protein degradation in stressed senescent barley (Hordeum vulgare cv. Proctor) leaves.

The molecular basis for the selectivity of protein degradation has been examined in osmotically stressed and senescent barley leaves. Relatively weak correlations between the in-vivo rate of loss of enzyme activity, and the charge and molecular weight of the enzymes ahve been detected. We interpret these correlations as supporting the view that the selectivity of enzyme degradation is the result of the physical properties of the enzymes being degraded. The weakness of the correlates is taken to mean that a number of properties which contribute to the selectivity are independent of one another. Under in-vitro conditions (autolysis at 0° C), the loss of enzyme activity was weakly correlated with the charge of the enzymes. However, there was a general similarity between the in-vivo pattern of loss of enzyme activity and the in-vitro patterns under a number of conditions. Furthermore double-isotope experiments demonstrated that the in-vivo degradation of soluble protein was reflected by in-vitro degradation under a number of conditions. Consequently we conclude that the selectivity of protein degradation is largely independent of the nature of the proteolytic system.