ALBERT

Notes: Abstract [32P]-labeled ATPase was isolated in a highly purified state fromMicrococcus lysodeikticus strain PNB grown in medium supplemented with [32P]orthophosphate. Selective extraction procedures allowed us to determine that at least 25% of the firmly bound label belonged to adenine nucleotides, ATP and ADP being present in equimolar amounts. However, no32P label was found to be part of phospholipids. This was confirmed by purification of the ATPase from cells fed with [2—3H]glycerol. Using the luciferin-luciferase assay we estimated that ATPase freshly isolated by Sephadex chromatography (specific activity 10–14 µmole substrate transformed · min−1 · mg protein−1) contained 2 moles ATP/mole of enzyme. The ratio fell with the age of enzyme and its purification by gel electrophoresis and this was paralleled by a loss of ATPase activity. The endogenous nucleotides were readily exchanged by added ADP or ATP. This result suggests that the sites for tight binding of adenine nucleotides are equivalent, although ADP seems to have a higher affinity for them. The last properties represent a peculiar characteristic of this bacterial ATPase as compared with other bacterial and organelle energy-transducing proteins.

Notes: Summary ‘High-affinity’ binding mechanisms for glycine exist in synaptosome-enriched preparations of various regions of rat CNS. Such mechanisms may represent interactions of glycine with its synaptic receptors.

Notes: Résumé On a étudié les possibilités de l'usage du photomètre de flamme pour la détermination quantitative de quelques éléments fréquents dans diverses alliages ferreuses. Parmi ces éléments on a étudié spécialment le cobalt, le nickel, le chrome et le manganèse. La sensibilité de ces éléments dans les méthodes de photométrie de flamme est petite et leur émissions sont affectées par la grande concentration du fer des échantillons, même dans les aciers avec des teneurs élevées de nickel et de chrome. On doit donc faire une séparation préalable du fer, que nous avons effectuée par extraction avec des solvants organiques, ayant étudié de même les récupérations quantitatives des autres métaux dans les solutions privées de fer. D'autre part on a étudié les interférences mutuelles de ces éléments pour arriver à des conclusions sur les erreurs que peuvent apparaître on cours des mesures. On discute aussi la séparation préalable du nickel et les modes opératoires. Quelques résultats sont donnés sur l'analyse de divers échantillons d'acier et de ferromanganèse. Les différences entre les valeurs photométriques et obtenues par voie chimique sont aussi indiquées.

Notes: Summary High-affinity, Na+-independent binding of β-alanine to a synaptosomal fraction of rat brain was potently inhibited by glycine and by some other α-amino acids, but not by taurine or GABA. This binding mechanism, which was also sensitive to both bicuculline and strychnine, might involve synaptic receptors for both β-alanine and glycine.

Notes: Summary Two new forms of the plasma membrane ATP-ase ofMicrococcus lysodeikticus NCTC 2665 were isolated from a sub-strain of the microorganism by polyacrylamide gel electrophoresis. One of them had a mol.wt of 368,000 and a very low specific activity (0.80 µ mol.min−1.mg protein−1) that could not be stimulated by trypsin. This form has been called BI (strain B, inactive). If the electrophoresis was carried out in the presence of reducing agents (i.e., dithiothreitol) and the pH of the effluent maintained at a value of 8.5 another form of the enzyme was obtained. This had a mol.wt of 385,000 and a specific activity of 2.5–5.0 µ mol.min−1.mg protein−1 that could be stimulated by trypsin to 5–10 µ mol.min−1.mg protein−1. This preparation of the ATPase has been called form BA (strain B, enzyme active). The subunit composition of both forms has been studied by sodium dodecyl sulphate and urea gel electrophoresis and compared to that of the enzyme previously purified from the original strain (form A). The three forms of the enzyme had similarβ and δ subunits, with mol.wt of about 50,000 and 30,000 dalton, respectively. They also had in common the component(s) of relative mobility 1.0, whose status as true subunit(s) of the enzyme remains yet to be established. However, subunitα, that had a mol.wt of about a 52,500 in form A (Andreu et al. Eur. J. Biochem. (1973) 37, 505–515), had a mol.wt similar toβ in form BI and about 60,000 in form BA. Furthermore BA usually showed two types of this subunit (α′ andα′′) and an additional peptide chain (ε) with a mol.wt of about 25,000 dalton. This latter subunit seemed to account for the stimulation by trypsin of form BA. Forms BA could be converted to BI by storage and freezing and thawing. Conventional protease activity could not be detected in any of the purified ATPase forms and addition of protease inhibitors to form BA failed to prevent its conversion to form BI. The low activity form (BI) was more stable than the active forms of the enzyme and also differed in its circular dichroism. These results show thatM. lysodeikticus ATPase can be isolated in several forms. Although these variations may be artifacts caused by the purification procedures, they provide model systems for understanding the structural and functional relationships of the enzyme and for drawing some speculations about its functionin vivo.

Notes: Summary Basal and trypsin-stimulated adenosine triphosphatase activities ofEscherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5mm, Vmax (minus trypsin) = 1.6µmol·min−1·mg protein−1, Vmax (plus trypsin) = 2.44µmol·min−1·mg protein−1; for the soluble ATPase: [S0.5] = 1.2mm, Vmax (-trypsin) = 4µmol·min−1·mg protein−1; Vmax (+trypsin) = 6.6µmol·min−1·mg protein−1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4mm) showed two sites (groups) with different K ms: at low ATP the values were 0.38 and 1.4mm for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20mm, respectively. Mg2+ saturation at constant ATP (8mm) revealed michaelian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulatesE. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors ofEscherichia coli ATPase. The Ki values for Pi were 1.6 ± 0.1mm for the membrane-bound ATPase and 1.3 ± 0.1mm for the enzyme in soluble form, the Ki values for ADP being 1.7mm and 0.75mm for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4mm) inhibited the membrane-bound enzyme by 60–70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20µ m) inhibited both states ofE. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.