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Sloane-Stanley (1953) and Rodnight (1956) described the hydrolysis of ox-brain 'diphosphoinositide' preparations (Folch, 1949) by extracts of brain and other tissues. Dawson (1954) and Hokin & Hokin (1956) showed that the phosphoinositide fraction of several tissues had a high tumover rate, as measured by the incorporation of 82P. The exact structure of the phosphoinositides involved is not known in every case, but it seems that the phosphate of phosphatidylinositol has a rapid tumover in several tissues (see Hawthorne, 1960a). In view of these observations a study has been made of the hydrolysis of phosphatidylinositol and 'diphosphoinositide' by extracts of animal tissues. With the latter compound, Rodnight (1956) observed enzymic release of both organic and inorganic phosphate. This suggested that more than one enzyme was involved in the degradation and stimulated the present attempt to purify the enzyme catalysing the primary hydrolytic step in the breakdown of phosphoinositides. Dawson (1959) has shown that phosphoipase B preparations from PeniciUium notatum and ox pancreas attack phosphatidylinositol. A preliminary report on this work has been published elsewhere (Kemp, Hubscher & Hawthorne, 1959). MATERIALS AND METHODS Analytical methods. The methods used for the determination of phosphate, nitrogen and glycerol have been described by Hawthorne & Hubscher (1959). Carboxylic ester was determined by the hydroxamate method of Stern & Shapiro (1953) and protein by the Gornal, Bardawill, & David (1949) modification of the biuret method. Where solutions were very dilute, the ratio of absorption at 260 and 280 m,u was used to measure the protein content. Paper chromatography. Methods used for the chromatography of glycerol, inositol, choline, ethanolamine and serine have been described before (HEbscher & Hawthorne, 1957). Inositol 1-phosphate and inositol 2-phosphate were separated by chromatography in propan-l-ol-aq. NH3 soln. (sp.gr. 0-88)-water (6:3: 1, by vol.) (Pizer & Ballou, 1959). Other solvents for the separation of inositol phosphate and * Part 2: Hawthorne, Kemp & Ellis (1960a).

1. Homogenates of the mucosa of the small intestine of the guinea pig were separated by fractional sedimentation into seven different fractions. The enzymic properties of some of these subcellular fractions were compared with those obtained from the mucosa of the small intestine of the rabbit and cat. 2. The enzymic properties of the low-speed sediment (15000g-min.) were investigated and it was shown that invertase and alkaline ribonuclease were predominantly located in this subcellular fraction, whereas alkaline phosphatase, aryl-amidase, acid phosphatase, acid ribonuclease and phosphoprotein phosphatase, though true constituents of this fraction, occurred to varying degrees in other subcellular structures also. 3. It was shown that the most probable source of the enzymic activities observed in the low-speed sediment was the brush border. Electron micrographs of the purified brush-border fraction indicated vesicles derived from the brush-border membrane. 4. A method is described for the fractionation of mucosal homogenates into a brush border-plus-nuclei fraction, a mitochondrial fraction, a microsomal fraction and a particle-free supernatant. The fractions were shown to be relatively pure, as indicated by the distribution of invertase, DNA, succinate dehydrogenase, glucose 6-phosphatase and 6-phosphogluconate dehydrogenase. 5. Most of the activity of four lysosomal enzymes present in the nuclei-free homogenate was sedimented at 375000g-min., suggesting the occurrence of lysosomal particles in mucosal homogenates. 6. Further fractionation of the microsomal membranes into three fractions is described. The enzymic composition of the membrane fractions is given and discussed in relation to their structure as seen in electron micrographs.

1. After conventional fractionation of rat liver homogenates in 0.88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.

Glyceride biosynthesis catalysed by the mitochondrial fraction of rat liver or by the microsoma1 fraction of cat intestinal mucosa is greatly stimulated by the supernatant fraction (6,000,000 x g x min). This stimulation is due to the presence of several factors and evidence is presented that one of these factors is a phosphatidate phosphohydrolase. The catalytic activities of the phosphatidate phosphohydrolases present in the mitochondrial and supernatant fractions were studied using as subxtrates either an aqueous dispersion of phosphatidate or membranebound phosphatidate formed as an intermediate in the biosynthesis of glycerides.The mitochondrial phosphatidate phosphohydrolase has a high activity with aqueous phosphatidate dispersions and a low activity with membrane-bound phosphatidate present as internal or added substrate. I n contrast, the phosphatidate phosphohydrolase of the supernatant fraction has a low activity with aqueous phosphatidate dispersions and a high activity with membranebound phosphatidate.As Mg++ and F-ions are usually included in the assay systems employed for measuring glyceride biosynthesis, the effect of these ions on the particulate and soluble phosphatidate phosphohydrolases was studied to exclude the possibility that the stimulation of glyceride biosynthesis by the phosphohydrolase of the supernatant fraction was due to an artefact. It is suggested that the phosphatidate phosphohydrolase isolated in the supernatant fraction is the major phosphohydrolase activity involved in glyceride biosynthesis.