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Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (chol) on chromosome V. The lipid composition suggests that chol mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the chol mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in chol cultures.

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The protein component of tissue thromboplastib (Factor III) from human brain was purified by extraction of a microsomal fraction with sodium deoxycholate, gel filtration of the extract on Sephadex G-100 and preparative polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The product, apoprotein III, was homogeneous by anayltical polyacrylamide-gel electrophoresis, and it induced monospecific antibodies in rabbits and goat as shown by immunodiffusion and immunoelectrophoresis. Amino acid- and carbohydrate-analysis data for apoprotein III are presented. The carbohydrate moiety of the protein consists of fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminate, amounting to a total content of 6.3g/100g. The apoprotein alone had no procoagulant activity. When Factor III was reconstituted by combining the pure apoprotein with a purified lipid fraction from the deoxycholate extract of crude Factor III, a high and optimal procoagulant activity was obtained at a phospholipid/protein ratio of 1.1g/g. Phosphatidylethanolamine alone had a weak but significant ability to restore activity, whereas phosphatidylcholine and phosphatidylserine separately had almost none. Two-component mixtures were on average more effective, and three-component mixtures far more effective, than the single phospholipids. The inclusion of a small amount of phosphatidylserine was very important for high activity.

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SummaryMonospecific antisera against the purified protein component of tissue thromboplastin (apoprotein-III) from human brain have been raised in goats and rabbits. The antisera neutralized tissue thromboplastin prepared from brain, thyroid gland and pulmonary tissue, indicating that apoproteins in the various preparations cross-reacted immunologically and therefore were similar or identical.Comparison of the activities of tissue thromboplastin preparations from 34 different areas of the brain demonstrated a characteristic distribution pattern and a wide range of activities. White and grey matter from the same areas had similar activities. Bulbus and tractus olfac-torius, medulla oblongata, corpus pineale, hippocampus and hypothalamus contained 160–270 % of the average activity, whereas cerebellum, globus pallidus, nucleus ruber and substantia nigra contained 30–60 %. The distinct distribution pattern was unrelated to tissue vascularization, and may suggest that apoprotein-III could serve other functions, apart from the coagulation of blood. The predominance in phylogenetically older brain regions would suggest that it represents a primitive or fundamental feature.

1. The effects of ATP, PP(i) and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ;magnesium' refers to total Mg(2+), both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the K(m) values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6.8mm in systems buffered with either tris-hydrochloric acid or glycylglycine-sodium hydroxide, but the K(m) values were different in these systems. The K(m) for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris-hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine-sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the K(m) values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris-hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10-20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25-50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg(2+) ion. 5. In the presence of 6.8mm-ATP no reaction occurred below 4-6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10-25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6.8mm-PP(i) or 6.8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6-8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (K(i) 2.1x10(-2)m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.