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The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata).

This study describes the entrapment of squid-type diisopropylphosphorofluoridate-hydrolyzing enzyme (DFPase) within mouse red blood cells. These erythrocytes thereby gain the ability to rapidly hydrolyze alkylphosphate cholinesterase (ChE) inhibitors such as diisopropyl fluorophosphate (DFP). DFPase rapidly hydrolyzes DFP to diisopropyl phosphate. Resealed erythrocytes provide a stable carrier system that can preserve the activity of encapsulated enzymes against otherwise rapid in vivo degradation; thus, ChE inhibitors can be degraded to relatively nontoxic metabolites by these erythrocyte carriers. Squid DFPase was purified from the hepatopancreas of Atlantic squid and DFPase activity was determined by measuring changes in fluoride ion concentration using a fluoride ion selective electrode. Mouse erythrocytes in suspension with excess squid DFPase were dialyzed against hypotonic buffer to allow the encapsulation of the enzyme to occur. Cells were then resealed by returning the suspension to isosmotic with saline. Rate of DFP hydrolysis observed with these cells was much greater than the rate of nonenzymatic hydrolysis and was directly proportional to the amount of the erythrocyte suspension added to the assay solution. The rate of hydrolysis was first order in substrate. Erythrocyte controls showed no endogenous DFPase activity. These results suggest that enzyme entrapment may be developed as a method to prevent and antagonize organophosphate poisoning.

Calcium-activated proteases have been purified from a number of vertebrate tissues, including the retina and lens. These proteases exhibit similar characteristics and are believed to be involved in the regulation of cytoskeletal elements. Here we report the partial purification and characterization of a calcium-activated protease from the squid photoreceptor cell which, when activated, specifically removes 10 kDa from the carboxyl-terminal of squid rhodopsin. No other detectable soluble proteins from the invertebrate photoreceptor are susceptible to cleavage and only one non-opsin, integral membrane protein shows evidence of cleavage. The enzyme requires 5 mM calcium for half maximal activation, and is not significantly activated by other divalent ions. The protease has a molecular weight of approximately 350 kDa, as determined by gel filtration, and when partially purified by casein affinity chromatography, it runs as three main bands of 76, 63 and 36 kDa on SDS-PAGE. The crude protease loses as much as 80% of its activity in 24 hr, whereas the partially purified protease is stable up to 4 weeks at 4 degrees C.

An enzyme in E. coli that hydrolyzes diisopropylphosphorofluoridate (DFP) has now been found to hydrolyze the nerve gas 1,2,2- trimethylpropylmethylphosphonofluoridate (soman) many times faster. With either substrate the E. coli enzyme is stimulated manyfold by 10(-3) M Mn2+. These criteria are combined and applied to this, and to a superficially similar but distinctly different, enzyme found in squid nerve. The results suggest that while several tissues of the squid contain only this second kind of DFP hydrolyzing enzyme, termed squid type DFPase , many other sources including E. coli contain a mixture of squid type DFPase (the name not strictly indicative of source) and the other DFP hydrolyzing enzyme, now termed Mazur type DFPase . Procedures for the purification of Mazur type DFPase from hog kidney, while increasing the specific activity for DFP hydrolysis may actually have been enriching the purified material in the squid type DFPase . Because E. coli has the highest soman hydrolyzing capacity of any source so far examined, this organism is a promising source for the development of new purification procedures for Mazur type DFPase .