Date of Award
Master of Science (MS)
B. Ann Oaks
'The enzymes responsible for hydrolysis of reserve protein in endosperm tissue during early growth of maize seedlings were investigated. The ma~n areas of research included:
(1) studies to optimize endopeptidase, carboxypeptidase and aminopeptidase reactions.
(2) a partial purification of the' endopeptidase protein.
(3) an investigation of the e~fects of certain plant hormones on the formation of the peptide hydrolase activities in en90sperm tissue of maize (W64 x W182E).
Assays which could measure endopeptidase, carboxypeptidase and aminopeptidase activities were studied in extracts of endosperm tissue prepared from Zea mays. As a substrate for the endopeptidase assay haemoglobin was more reactive than gliadin, BSA, azocoll or azocasein. Synthetic substrates were used to assay carboxypeptidase activity, due to Ala) and aminopeptidase (Leu-para-nitroaniline). Interference with measurement of carboxypeptidase activity, due to autolysis, could be eliminated by dialysis of the extract. The dependence of the activities upon pH, concentration of substrate, volume of extract and temperature were investigated. Optimum conditions were established. Routinely, saturating protein concentrations were used in the acidic endopeptidase assays whereas the peptidase assays were performed at less than saturating substrate concentrations due to limited solubility of the substrates.
The effects of selected enzyme inhibitors on the enzyme activities were tested. Para-chloromercuribenzoate gave the greatest inhibition of all activities. Aminopeptidase was most sensitive, followed by carboxypeptidase and then acid endopeptidase. Other inhib~tors (PMSF and EDTA) had slight effects. Aminopeptidase was sensitive to 5 mM phenylmethylsulphonylfluoride whereas the carboxy-peptidase and endopeptidase were not significantly affected.
A number of techniques were used in an attempt to purify the acid endopeptidase. Acid endopeptidase activity was clearly separated from carboxypeptidase activity on Sephadex G-50. The best partial purification of an endopeptidase fraction was achieved with Sephadex G-50 chromatography, followed by CM-cellulose chromatography. A thirteen fold increase in specific activity was attained. The molecular weight of the most pure fraction was estimated to be 21,000 daltons, using a Sephadex G-200 column.
The development of acid endopeptidase, carboxypeptidase and aminopeptidase was followed in the post pollination and post germination periods. The exopeptidase activities were high in the post pollination tissues, while acid endopeptidase was low. The latter activity rose after imbibition, reaching a peak at five to eight days. Carboxy-peptidase also increased in activity following imbibition, while aminopeptidase activity declined from the value found in the mature caryopsis.
Endosperms, from which the embryo and scutellum had been removed were incubated in the presence of plant hormones and chemical effectors. Unlike barley (Hordeum vulgare), in which GA₃ is required to induce development of a protease activity in halfseeds, GA₃ is not required for development of endopeptidase activity in deembryonated caryopses of maize (Zea mays). In the present study, it was found that GA₃ did not affect the appearance of exopeptidase activities either. Incubation with 2 μM abscisic acid (ABA) caused partial inhibition of the development of endopeptidase and carboxypeptidase activities. At a concentration of 10 μM, significant inhibition of aminopeptidase develppment was also seen. GA₃ (30 μM) could partially reverse the inhibition of acid endopeptidase and carboxypeptidase caused by the lower concentration of ABA. Cordycepin, an inhibitor of RNA formation, was found to have no effect on either the development of protease activity, or the inhibition brought about the ABA, although it did inhibit development of α-amylase in barley and maize endosperm pieces. Cycloheximide, a protein synthesis inhibitor drastically decreased the production of all peptide hydrolase activities.
Winspear, Michael J., "Peptide Hydrolases in Maize Endosperm" (1981). Open Access Dissertations and Theses. Paper 4036.