Spinach chloroplasts were purified on gradients of Percoll which preserved envelope impermeability and CO2-dependent oxygen evolution in the light. Application of (35)SO4″ to purified chloroplasts resulted in a light-dependent labeling of a lipid component which was indentified as sulfoquinovosyl diacylglycerol. Fractionation of chloroplasts showed that after 5 min of labeling most of the newly synthesized sulfolipid was present in thylakoids. Only a small percentage was recovered from the envelopes. Molecular species from envelopes and thylakoids were identical. The molecular species did not change during incubation times ranging from 5 min up to 4.5 h. Mesophyll protoplasts from (35)SO4″-labeled oat primary leaves were gently disrupted and separated into organelles by sucrose gradient centrifugation. Labeled sulfolipid was located almost exclusively in the chloroplasts. This, in combination with the experiments carried out with isolated chloroplasts, indicates that the final assembly steps in the biosynthesis of sulfolipid are confined to the chloroplasts.

Vacuoles were released from oat (Avena sativa) mesophyll protoplasts and purified by sedi­mentation and flotation. Disruption of isolated vacuoles followed by density gradient centri­fugation gave two membrane bands which after combination were further purified on sucrose gradients. A significant contamination by microbodies, thylakoids, mitochondria, endoplasmic reticulum and Golgi membranes can be excluded, whereas markers for plasma membrane and chloroplast envelope were present in the final membrane preparation.In the purified membrane fraction the following enzymatic activities were detected: NADH- cytochrome C reductase E.C. 1.6.2.2, ATPase E.C. 3.6.1.3. UDPG: sterol glucosyltransferase, UDP-Gal: diacylglycerol galactosyltransferase E.C. 2.4.1.46, glucan synthetase II, CDP-choline: diacylglycerol phosphocholinetransferase E.C. 2.7.8.2, formation of acylgalactosyl diacylglycerol and acyl-CoA thioesterase E.C. 3.1.2.2. None of these can be considered to be specific for the tonoplast. Acid phosphatase E.C. 3.1.3.2 was present in the cell sap.The vacuolar membranes contain phospholipids and glycolipids of the most complex composi­tion found so far in a membrane system isolated from mesophyll protoplasts. About half of the glycolipids were accounted for by glycosyl diacylglycerols usually considered to be confined to plastids. Steryl glycosides and acyl steryl glycosides were other prominent glycolipids. A cerebroside was the predominating lipid component of this membrane preparation.

De novo-synthesis of glycerolipids in chloroplasts is initiated by a stroma enzyme which catalyzes the formation of lyso-phosphatidic acid from glycerophosphate and acyl-CoA. When these substrates are added to isolated, intact chloroplasts, only glycerophosphate can readily pass through the chloroplast envelope which represents a permeation barrier for acyl-CoA, although higher thioester concentrations destroy this membrane system. At low concentrations of acyl-CoA, which do not impair the envelope, intact chloroplasts metabolize exogenous acyl-CoA in two ways to give free fatty acids and labelled phosphatidyl choline. This indicates that the envelope thioesterase can use exogenous substrates. Isolated, intact chloroplasts fixing radioactive CO2 label free fatty acids and acylglycerols but not galactolipids, since they cannot convert 3-phosphoglycerate into UDP-galactose which in vivo is supplied by the cytoplasm. This cooperation was simulated in vitro by adding all enzymes and cofactors necessary for conversion of 3-phosphoglycerate into UDP-galactose to intact chloro­plasts which then formed labelled monogalactosyl diacylglycerol from labelled CO2. The time required to transfer envelope-made galactolipids from the envelope into thylakoids was studied by incubating intact chloroplasts with radioactive UDP-galactose, subsequent osmotic disruption of organelles with concomitant enzymatic degradation of UDP-galactose followed by separation of envelopes and thylakoids. Only after short times (< 1min) appreciable proportions 920-30%) of radioactive galactolipid export from envelopes into thylakoids.

Mitochondria were isolated from oat primary leaves via mesophyll protoplasts and subjected to phospholipid analysis. In mesophyll cells mitochondria account for only small proportions of cellular phospholipids (in the order of 5%) and proteins (in the order of 2%). Contamination by lipids from other membranes was insignificant as indicated by the absence or very low levels of chlorophyll, galactolipids and steryl glycosides. The absence of 3-iraws-hexadecenoic acid in phosphatidylglycerol from mitochondria of green cells serves as an additional criterion of purity. The phospholipid mixture extracted from these mitochondria resembles phospholipids in mitochondria from non-green tissues regarding composition as well as fatty acid profiles. Therefore, mitochondria maintain a rather constant lipid profile and in contrast to plastids do not respond at this level to differences in the physiological status of their housing cell. Palmitic acid in mitochondrial phosphatidylcholine and phosphatidylethanolamine is primarily localized at the C-l position of the glycerol moiety. Two enzymatic activities so far not described in mitochondria, formation of acylgalactosyl diacylglycerol and hydrolysis of acyl-CoA, were found in the purified mitochondrial fraction.