The precursor of interleukin-1 alpha is phosphorylated at residue serine 90

Mononuclear phagocytes release interleukin-1 (IL-1), a 17-kDa polypeptide with diverse biological activities. IL-1 is synthesized as a precursor (31 kDa) which lacks a signal sequence or hydrophobic domains that could facilitate transmembrane translocation. Possible postsynthetic modifications of IL-1 that might account for its cellular transport were examined. We found that lipopolysaccharide stimulated, but not unstimulated, murine macrophages incorporated 32PO4 into the IL-1 alpha precursor (31 kDa) predominantly at residue serine 90. Released IL-1 alpha (17 kDa) is not phosphorylated in agreement with peptide sequence data that the site of 32P incorporation is in the amino-terminal one-third of the precursor. Approximately 10% of the phosphorylated IL-1 alpha precursor is membrane bound and associated with a fraction enriched in lysosomal vesicles. Together these data suggest mechanisms by which the postsynthetic proteolysis of the IL-1 alpha precursor may be modified and cellular transport of IL-1 alpha is accomplished.     

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.     

Separation of Legionella pneumophila proteases and purification of a protease which produces lesions like those of Legionnaires' disease in guinea pig lung

Six discrete protease activities were recovered from the supernatant broth of Legionella pneumophila cultures by ion-exchange chromatography. One of these demonstrated in vitro activity against collagen, casein and gelatin. When administered into the lungs of guinea-pigs this protease elicited lesions which were pathologically similar to those seen in clinical and experimentally induced Legionnaires' disease.     

Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.     

Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam: kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate

The kinetics of NADH consumption, oxygen uptake, and hydrogen peroxide production have been studied for norcamphor metabolism by cytochrome P-450cam. The kinetic deuterium isotope effects on these processes, with specifically deuteriated norcamphor, are 0.77, 1.22, and 1.16, respectively. Steady-state UV-visible spectroscopy indicates that transfer of the second electron to the dioxy ferrous P-450 is the rate-limiting step, as it is when camphor is the substrate. The inverse deuterium isotope effect for NADH consumption is consistent with an isotope-dependent branching between monooxygenase and oxidase activity, where these reactivities differ in their NADH:oxygen stoichiometries. However, no isotope-dependent redistribution of steady-state intermediates was detected by isotopic difference UV-visible spectroscopy in the presence of norcamphor. The kinetic isotope effects and steady-state spectral results suggest that the high-valent iron-oxo hydroxylating intermediate [FeO]3+ is reduced by NADH and the physiological electron-transfer proteins to afford water.