Citrate-tris(hydroxymethyl)aminomethane-mediated release of outer membrane sections from the cell envelope of a deep-rough (heptose-deficient lipopolysaccharide) strain of Escherichia coli O8.

A heptose-deficient lipopolysaccharide strain of Escherichia coli O8, strain F515, was found to release portions of its outer membrane when cells were exposed to 10 mM citrate buffer (pH 2.75) for 30 min and subsequently exposed to 100 mM tris(hydroxymethyl)aminomethane buffer (pH 8.00). The outer membrane component release was found to be composed of protein, lipopolysaccharide, phospholipid (cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol), and alkaline phosphatase. The outer membrane component was released from the cell envelope in the absence of cell lysis, as no glucose-6-phosphate dehydrogenase activity or succinic dehydrogenase activity was detected. Morphologically, the outer membrane component appeared to consist of laminar fragments and vesicles which had an associated alkaline phosphatase activity.     

Purification of native forms of eel acetylcholinesterase: active site determination

The 14 and 18 S forms of acetylcholinesterase from the electric organ of Electrophorus electricus were purified by chromatography on an N-methyl-3-aminopyridinium derivative of Affi-Gel 202. a further increase in purity was seen when these forms were separated by density gradient sedimentation subsequent to the affinity step. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated that the 14 and 18 S forms were highly purified following these procedures. Using [³H]diisopropyl fluorophosphate labeling and separation of labeled enzyme from unreacted [³H]diisopropyl fluorophosphate by gel filtration, active site numbers of 8.3 and 11.4 were determined for the 14 and 18 S forms, respectively. These numbers compare to 4.2 active sites determined for the 11.8 S globular form of acetylcholinesterase. These results are in accord with a proposed model of two and three tetrameric structures comprising the head groups of the 14 and 18 S forms of electric tissue acetylcholinesterase.     

Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe.

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.