The chromosomally mediated penicillinase present in three strains of Escherichia coli K-12 has been purified and characterized. Two of the strains carried the ampA gene and the third the wild-type allele. The purification involves release of the enzyme by spheroplast formation, dialysis, chromatography on sulfoethyl cellulose, and chromatography on hydroxylapatite. Enzyme from the two mutants appeared homogeneous in polyacrylamide gel electrophoresis. Enzyme from the wild-type strain gave two bands. Immunologically, the enzymes from all three strains were identical. Ultracentrifugation gave a homogeneous peak with a sedimentation coefficient of 3.45. Gel filtration gave an estimated molecular weight of29,000. The Nterminal amino acid residue was found to be alanine. Complete amino acid analysis showed a lack of cysteine. Ultraviolet spectra were recorded at three different pH values. The extinction coefficient at 280 nm is 21.0 for a 1% solution at pH 6.8. The optimal pH is 7.3. With enzyme from one of the resistant mutants, the following Km and turnover number values were obtained: for penicillin G, 12 ;uM and 2,080; for D-MATERIALS AND METHODS Bacterial strains. All strains used were E. coli K-12. Strain Gllal which carries the amp Al allele is a spontaneous mutant obtained from the wild-type strain Gll (10). The ampAl allele was transduced into strain D2, which afterwards was mutated to the highly ampicillin-resistant strain D3 (5). A second mutation step induced streptomycin resistance in D3, thus producing strain D31 (5). Strains Gllal and D2 both form single cell colonies on plates containing D-ampicillin concentrations of 15 to 20,g/ml, whereas strain D31 can form single-cell colonies on plates with D-ampicillin concentrations of 75 to 100 ,Ag/ml (for resistance determinations, see references 6 and 24). Media and growth conditions. The growth medium 218

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Carefully washed human platelets contain calcium in an unusual state of chemical combination. Platelet calcium is non-ionic and is not exchangeable with ionic Ca45. On extraction of platelets with lipid solvents, the calcium separates with the phospholipids. The calcium-lipid complex thus obtained does not undergo any exchange with radioactive calcium. Study of other properties of platelets reveals that the clot-promoting activity of platelets (activated by antihemophilic factor and plasma thromboplastin component) also separates into the lipid fraction. Preliminary observations are reported on the nature of platelet lipids as revealed by paper chromatography and paper electrophoresis in solvents of low dielectric constant. Small amounts of proteolipid have been isolated from the lipid extracts. The unique calciumlipid complex is thought to originate in the cell membrane of the platelet.

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SummaryStudy of the kinetic behavior of purified human clotting factors suggests that formation of prothrombin activator from platelets, plasma thromboplastin component (PTC), antihemophilic factor (AHF), Factor V and calcium proceeds by a sequence of reactions. The earliest detectable reaction is slow and involves platelets, PTC and calcium; an intermediate activated PTC is obtained which is free of platelet contamination and is characterized by its kinetic behavior. Platelets are also requisite at a later step in the sequence, possibly in an intermediate formed between activated PTC, AHF, platelets and calcium. Variations in platelets, AHF and PTC result in characteristic responses in the kinetics of thrombin formation.