The epoxide hydrolase from Aspergillus niger was purified to homogeneity using a four-step procedure and p-nitrostyrene oxide (pNSO) as substrate. The enzyme was purified 246-fold with 4% activity yield. The protein is a tetramer composed of four identical subunits of molecular mass 45 kDa. Maximum activity was observed at 40 8C, pH 7.0, and with dimethylformamide as cosolvent to dissolve pNSO. Hydrolysis of pNSO was highly enantioselective, with an E value (i.e. enantiomeric ratio) of 40 and a high regioselectivity (97%) for the less hindered carbon atom of the epoxide. This enzyme may be a good biocatalyst for the preparation of enantiopure epoxides or diols.

A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 x g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-l,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-l,5-bisphosphate carboxylase/ oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.Many diverse physiological processes that occur in plants in response to light, growth regulators or to a stress are mediated via protein phosphorylation 11-31. Membranebound protein kinases, but also soluble ones, have been described in plant tissue 12, 4, 51.In chloroplasts, phosphorylation of proteins occurs mainly in thylakoids (on the light-harvesting chlorophyll-a/bbinding complex and on the N,N-dicyclohexylcarbodiimidebinding proteolipid) or in the chloroplast envelope [6 -lo]. To date polypeptide phosphorylated in the stroma of chloroplasts and soluble protein kinase have been poorly identified. Suss [l 11 has reported that light-exposed Vicia faba leaves incorporate 32P into a polypeptide comigrating with the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) after SDS/urea gel electrophoresis. But this author concluded that this polypeptide was not the large subunit because an immunoassay was negative. Recently another observation of phosphorylation of a polypeptide comigrating with the large subunit has been reported by Foyer [12] in spinach chloroplasts incubated with [32P]orthophosphate.In the present study we give evidence for the presence of a soluble chloroplast protein kinase able to phosphorylate stromal polypeptides two of them being the large subunit of Rubisco and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as demonstrated by immunological techniques. Some of the characteristics of large subunit phosphorylation are studied in vitro. MATERIALS AND METHODS Materials and reagentsSpinach plants (Spinaciu oleracea, var. Giant d'hiver) were grown on soil in a controlled room in the conditions already described [13] and were harvested after one month. Biochemicals were purchased from Sigma; RNasin, a RNase inhibitor, was from Genofit (Geneve); Percoll, Sephadex G-50, and protein-A-Sepharose CL-4B were from Pharmacia; Sorbitol was purified with mixed-bed resin (Bio-Rad AG 501-X8) and charcoal; [35S]methionine (43.3 TBq/mmol) ...

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