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The preparation from Percoll-purified cucumber (Cucumis sativus)etiochloroplasts of a subplastidic membrane fraction that is capable of high rates of Mg insertion into protoporphyrin IX is described. The plastid stroma was inactive when used either alone or in combination with the membrane fraction. Successful preparation of the subplastidic membrane fraction required that Mgprotoporphyrin chelatase was first stabilized by its substrate. This was achieved by lysing Percoll-purified plastids in a fortified hypotonic medium containing protoporphyrin IX prior to ultracentrifugation and separation of the stroma from the plastid membranes. Protoporphyrin IX became membrane bound. Other additives needed for enzyme activity fell into two groups: (a) those needed for enzyme stabilization during membrane preparation and (b) those involved in the primary mechanism of Mg insertion into protoporphyrin IX. Ethylenediaminetetraacetate belonged to the first group, magnesium belonged to the second group, and ATP belonged to both groups. specificity (21), and the role of ATP in Mg chelation has been hindered by the inability to purify the enzyme. Indeed, attempts at rupturing the plastids, a prerequisite for Mg-Proto chelatase purification, resulted invariably in loss of enzyme activity (4,15,22).While this manuscript was in preparation, Walker and Weinstein described a subplastidic system that overcomes the requirement of plastid intactness for Mg-Proto chelatase activity (23). The system was prepared from lysed pea (Pisum sativum) chloroplasts and consists of soluble and membranebound fractions. Attempts at preparing similar systems from cucumber chloroplasts were not successful (23). In the present work, we describe the preparation from cucumber etiochloroplasts of a stabilized subplastidic membrane fraction that is capable of high rates of Mg insertion into exogenous Proto without addition of a soluble stromal fraction. MATERIALS AND METHODS Plant Materials Preparation of Etiochloroplast Stroma and MembranesThe membrane fraction was separated from the stromal fraction after lysis of the Percoll-purified plastids. Lysis was achieved by addition to the pelleted Percoll-purified plastids (about 15 mg of protein) of 5 mL of lysing buffer. The plastid membranes were suspended to homogeneity at 40C with a small paintbrush. To stabilize the Mg-Proto chelatase activity, and unless otherwise indicated, 100 nmol Proto/0.33 mL of membrane suspension was added to the lysed plastids immediately after lysis. The stroma and membranes fractions were then resolved following ultracentrifugation at 235,000g for 1 h in a Beckman 80 Ti angled rotor at 10C (9). Assessment of the Extent of Plastid LysisThe extent of plastid lysis was determined from the activity of G6PDH (EC 1.1.1.44), a plastid stroma marker, in etiochloroplasts lysed by osmotic shock or by 0.1% Triton X-100. The G6PDH activity of Triton X-100-treated plastids was considered to represent 100% lysis. To measure G6PDH activity, 0.1-mL aliquots of lysed plastids were added ...

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Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.