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-p- Gas exchange between a leaf and the atmosphere is almost exclusively through stomata in the epidermis. Stomatal aperture is varied to minimize H20 loss while admitting CO2. The physical basis for opening is swelling of the surrounding guard cell pair in response to the large negative osmotic potential resulting from K+ influx (1). Potassium uptake is electrically balanced by Cl-uptake (2) and the synthesis of organic anions from starch (3-6).An (9) or as by Outlaw and Manchester (6). Analytical enzymes were from Boehringer except RuP2 carboxylase and ribose-P isomerase, which were from Sigma. Agarose was from Miles; most other chemicals were from Sigma. Microscopy Guard cells in epidermal peels were examined by brightfield and fluorescence microscopy. Filters for fluorescence studies were Leitz BG12 and K580. For transmission electron microscopy, tissue was fixed at room temperature for 2 hr under partial vacuum in 100 mM Na cacodylate (pH 7) containing 3% (wt/wt) glutaraldehyde. The tissue was postfixed for 2 hr with 1% OS04 in the above buffer and then dehydrated in a graded ethanol series. The alcohol was replaced by propylene oxide before the tissue was embedded in Spurr's resin (10). Thin sections were stained with uranyl acetate and lead citrate (11) and then were examined in a Hitachi HU-11C electron microscope operated at 75 kV.Enzyme assays Leaflets were illuminated (200 microeinsteins m-2 s-1 of 400-to 700-nm radiation) for at least 15 min before quenching in liquid N2 that had been reduced to its freezing point by evacuation. [Illumination was to activate the enzymes (12)(13)(14).] Leaflets were then freeze-dried at -35°C. Freeze-drying did not change the activities of any of the enzymes reported here.Samples were dissected and weighed (5-15 ng) on a quartz fiber balance in a room with controlled temperature and humidity. The early analytical steps were conducted in a small droplet under oil to prevent evaporation. All enzyme activities were measured at 230C, with the reaction initiated by addition of the tissue sample through the oil. Acid was used to destroy NAD(P)H before enzymatic amplification of the oxidized pyridine nucleotide (NAD, ref. 15; NADP, ref. 16). Tissue blanks and standards were carried through all steps. Except as noted, enzyme specific activities reported are based on dry weight. The concentration of most substrates was determined Abbreviation: RuP2, ribulose bisphosphate.

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 activitv 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.

The properties of homoserine dehydrogenase (EC 1.1.1.3) isolated from shoots of young etiolated seedlngs of Zea mays L. var. earliking can be reversibly altered by dialysis against an appropriate buffer. Treatment with 500 milimolar potassium pbosphate buffer (pH 7.5) in the absence of Lthreonine results in diminished regulatory control such that the enzyme becomes less sensitive to feedback inhibition. The physical and regulatory properties of experimentaliy altered and unaltered enzymes are compared with those of enzyme isolated from shoots of older seeings. 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 seedlngs of different ages is proposed. The initialiy 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.

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