Endopeptidase and Carboxypeptidase Enzymes of Vacuoles Prepared from Mesophyll Protoplasts of the Primary Leaf of Wheat Seedlings

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Summary

Vacuoles prepared from mesophyll protoplasts of the primary leaf of wheat (Triticum aestivum L. cv. Egret) contain carboxypeptidase (CP) and endopeptidase (EP) activity. CP activity was optimal at pH 5.2. The most preferred substrate was Z-Phe-Ala and activity against a wide range of other substrates was dependent on the C-terminal amino acid. Z-dipeptides with proline in any position were not hydrolysed. CP activity was inhibited by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, sodium dodecyl sulfate (SDS) and 4-hydroxymercuric benzoic acid. Thus the CP enzyme from the wheat leaf is a Serine Carboxypeptidase (EC 3.4.16).

EP activity, assessed by following the degradation of the Large Sub Unit (Mr 53.1 kD) of Ribulose 1,5-Bisphosphate Carboxylase (RuBPCase) by SDS-PAGE, was optimal at pH 5.2 (-SDS) and 5.5 (+SDS). The overall rate of degradation of the Large Sub Unit (LSU) was increased about 2.6-fold by the addition of 0.2 % SDS to the assay medium. In the absence of SDS there were only three prominent polypeptide breakdown products, Mr 52.1, 50.9 and 50.9 kD. The 50.9 and 50.1 kD products appeared resistant to further hydrolysis, whilst the 52.1 kD product was eventually degraded. The presence of SDS increased the heterogeneity of the breakdown products, polypeptides with Mr 51.8, 44.4, 43.6, 30.3 and 29.5 kD were especially prominent.

The major portion of the vacuolar EP activity was considered to belong to the Thiol proteinase group (EC 3.4.22), although indirect evidence was obtained for two other EP activities. There appeared to be little co-operation between the EP and CP activities in the degradation of RuBPCase.

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