Comparison of sensitive and desensitized forms of maize homoserine dehydrogenase.

The properties of homoserine dehydrogenase (EC 1.1.1.3) isolated from shoots of young etiolated seedlings of Zea mays L. var. earliking can be reversibly altered by dialysis against an appropriate buffer. Treatment with 500 millimolar potassium phosphate buffer (pH 7.5) in the absence of l-threonine results in diminished regulatory control such that the enzyme becomes less sensitive to feedback inhibition. The physical and regulatory properties of experimentally altered and unaltered enzymes are compared with those of enzyme isolated from shoots of older seedlings. Multiple forms of both sensitive and insensitive enzymes are identified, and a model which is consistent with the observed isozymes and the difference in regulatory properties of enzymes obtained from seedlings of different ages is proposed. The initially sensitive enzyme is postulated to undergo a conformational change followed by formation of insensitive multimeric aggregated forms. The experimental conditions which facilitate alteration of the enzyme are discussed in relation to conditions which could occur in vivo.

Photosynthetic carbon reduction pathway is absent in chloroplasts of Vicia faba guard cells.

Four cell types from Vicia faba Linnaeus "Long Pod" leaflets were assayed for three enzymes unique to the photosynthetic carbon reduction pathway. The enzymes were ribulosebisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], phosphoribulokinase (ATP:D-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19), and glyceraldehyde-phosphate dehydrogenase (NADP(+)) (phosphorylating) [D-glyceraldehyde-3-phosphate:NADP(+) oxidoreductase (phosphorylating), EC 1.2.1.13]. On a dry weight basis, these enzyme activities were about twice as high in palisade as in spongy parenchyma. Two of the enzymes were not detected in epidermal cells and the other was present in only a trace amount. In guard cells, these enzyme activities were absent or present at les than 1% of the amount in palisade cells. Immunoelectrophoresis showed that ribulosebisphosphate carboxylase was absent in extracts of guard cell protoplasts. Microscopy confirmed the abundance of typical guard cell chloroplasts. These results demonstrate the absence of the photosynthetic carbon reduction pathway in guard cell chloroplasts. This is the only chloroplast type known to be deficient in this pathway in plants whose primary CO(2) acceptor is ribulose bisphosphate. Possible reasons for the absence of this pathway in guard cells are discussed.

Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase.

Properties of phosphoenolpyruvate carboxylase in guard cells dissected from frozen-dried Vicia faba L. leaflets were studied using quantitative histochemical techniques. Control experiments with palisade cells and whole leaflet extract proved that the single cell approach was valid. Most characteristics of enzyme activity in guard cells were identical to those in the leaflet extract. The activities were highly dependent on temperature, with maximum activity at 25 to 35 C. Half-maximum activity (with 1 millimolar phosphoenolpyruvate [PEP]) was observed at 0.1 millimolar Mg(2+). Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent K(m(PEP)) was 0.15 millimolar at pH 7.7; with guard cells, the values were 1.49, 0.5 to 0.8, and 0.24 millimolar in three successive experiments. Additional experiments showed that apparent K(m(PEP)) of guard cell activity from plants within a single growth lot was reproducible and did not change during stomatal opening. Mixed extract experiments proved that soluble compounds were not responsible for the difference observed between leaflet and guard cell activities. The differences in apparent K(m(PEP)) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed.

Changes in Enzyme Regulation during Growth of Maize: II. Relationships among Multiple Molecular Forms of Homoserine Dehydrogenase.

The relative contribution of each of several forms of homoserine dehydrogenase (EC 1.1.1.3) to the total enzyme population in etiolated shoots and in roots of Zea mays L. var. earliking was examined by the use of gel filtration chromatography and disc gel electrophoresis. In enzyme preparations derived from shoots of seedlings grown for 72, 120, or 168 hours, two molecular forms, II and III, which have the same apparent molecular weight but differ in net charge, contributed 75 to 80% of the total enzyme activity. A lower molecular weight species, form I, contributed 20 to 25% of the activity from 72-hour shoots, but was found to decrease concomitantly with a proportional increase in activity contributed by aggregated enzyme form(s) during shoot development. Form I contributed a comparatively larger fraction of the total enzyme activity in preparations of roots of 72-hour seedlings.The characteristic enzyme activity of different tissues was found to be the result of variations in both the amount and the properties of individual forms. Form I was consistently insensitive to inhibition by the feed-back modifier, l-threonine, but evidence is presented which indicates that the regulatory properties of form II and/or form III are systematically altered during shoot growth. The activity of the enzyme forms was also differentially stimulated by monovalent cations, K(+) being the most effective activator; in all cases the potential for activation was correlated with the potential for inhibition. In contrast to these differences among the forms of the maize enzyme, all forms were shown to share a number of common characteristics. Potential factors which could influence the growth-associated changes in homoserine dehydrogenase are discussed briefly.