Fragmentation of a mollusc haemocyanin with plasmin and immunological identification of the fragments.

Haemocyanin from the gastropod mollusc Lymnaea stagnalis (pond snail) was partially digested with plasmin under a variety of experimental conditions and the products of digestion analysed by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Fragments were obtained corresponding to one, two, three, four and five oxygen-binding domains, one domain having a mol.wt. of approx. 50000 and containing 2 ions of Cu. The fragments obtained after extensive digestion were non-identical immunologically, and summation of their molecular weights allowed a minimal mol.wt. of 413000 to be calculated for the original, undigested, eight-domain polypeptide chain. The use of mild-digestion conditions allowed the time course and sequence of the digestion to be monitored. An initial cleavage gave a three-domain and a five-domain fragment. The three-domain fragment was resistant to further digestion. The five-domain fragment could be digested further to give, successively a four-domain, a three-domain, and finally a two-domain fragment, single-domain units being cleaved. These data form the basis for a proposed sequence for the different domains in the original chain.

NADP-dependent isocitrate dehydrogenase from the mussel Mytilus edulis L. 1. Purification and characterisation.

A purification scheme is described for NADP-dependent isocitrate dehydrogenase from the digestive gland of Mytilus edulis. The scheme incorporates three chromatographic steps: hydroxyapatite adsorption, blue-Sepharose affinity chromatography and DEAE-Sephacel ion-exchange chromatography. The subunit molecular weight of the enzyme was 45 000 (+/- 5000) by sodium dodecylsulfate gel electrophoresis. The purified enzyme was homogeneous on dodecylsulfate gels and had a specific activity of 30--50 U/mg protein.

NADP-dependent isocitrate dehydrogenase from the mussel Mytilus edulis L. 2. Kinetic studies.

The kinetic mechanism for NADP-dependent isocitrate dehydrogenase from the digestive gland of the mussel Mytilus edulis has been investigated. Initial-rate studies and substrate-analogue and product-inhibition patterns are consistent with a rapid-equilibrium random-ordered reaction mechanism, both with respect to substrate addition and product release. Product inhibition by NADPH is non-competitive versus D-isocitrate concentrations. 2-Oxoglutarate inhibition is competitive versus D-isocitrate at all NADP concentrations. The inhibition by 2-oxoglutarate versus NADP is either non-competitive or uncompetitive. Tricarballylic acid (a substrate analogue of D-isocitric acid) gives competitive inhibition versus D-isocitrate at all NADP levels and mixed inhibition versus NADP, at sub-saturating levels of D-isocitrate.