Pig liver pyruvate carboxylase. Purification, properties and cation specificity

1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris(+) or triethanolamine(+). 8. The activity of the enzyme is dependent on the presence of acetyl-CoA; the low rate in the absence of added acetyl-CoA is not due to an enzyme-bound acyl-CoA. The dissociation constant for enzyme-bound acetyl-CoA is a marked function of pH.     

Cultivation and Survival Studies of Neisseria gonorrhoeae in a Human Diploid Cell Strain

Neisseria gonorrhoeae was cultivated in a human diploid cell strain (WI-38). Eighty percent of the cultures contained viable gonococci for at least 4 m at 36°C, as evidenced by subculture to brain heart infusion broth. Monthly subcultures of bacteria could be made to fresh WI-38 cultures for at least 11 monthly passages with a 69% survival rate. The identity of gonococci was confirmed by morphology, gram staining, oxidase testing and fermentation reactions. Viability in brain heart infusion broth, minimum essential medium (Eagle), and WI-38 spent fluid was of much shorter duration. The organisms grown in WI-38 cultures appeared to orient largely in the vicinity of the WI-38 cells as well as within the cytoplasm of the cells.     

Gallium-67 Scans in the Diagnosis of Pyelonephritis

Gallium-67 (⁶⁷Ga) citrate was administered intravenously (50 microcuries per kg of body weight) to patients in whom acute and chronic urinary tract infections were suspected. Scanning was done, using both the Anger-type scintillation camera and the rectilinear scanner, 24 to 78 hours after injection of the isotope.The preliminary results imply that ⁶⁷Ga renal uptake is present in patients with pyelonephritis whether overt or silent, as well as in patients with uretero-sigmoidostomies. However, ⁶⁷Ga renal uptake is not present in patients with radiographic evidence of chronic pyelonephritis without active infection and in patients without renal disease.     

Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP(2-) binds next, followed by HCO(3) (-) and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of P(i) and MgADP(-). 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][(32)P]P(i) equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, P(i), pyruvate and oxaloacetate.     

Properties of adenovirus messenger ribonucleic Acid synthesized in vitro

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 μg of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 μg of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.     

CAMBIAL DIVISIONS IN PSEUDOTSUGA MENZIESH

Mitotic activity in the vascular cambium was determined from nine samples from a single internode in each of four Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) trees. Counts of mitotic nuclei and populations of potentially dividing cells in each sample were used to determine the average mitotic index per core. The sampling error was determined for the average mitotic index per core and the internode as a whole. Significant differences were found between the mitotic indexes of samples within the internodes of three of the trees; however, no differences were observed in the rate of division among trees. A significant correlation was established between the number of cells in the cambial zone and the average mitoses per core per sample.     

Microelectrode Studies of Dog's Gastric Mucosa

In anesthetized dogs, the potentials in the mucous coat and gastic cells were measured with microelectrodes. In the secreting stomach, with isotonic saline in contact with the mucosal surface, the orientation of the initial change in potential difference (PD) was often the same as that of the liquid junction potential between gastric juice and saline (the microelectrode became negative to a reference electrode in the saline) but the magnitude of the change was never more than 11 mv. On the basis of this finding an explanation is offered for the observation that in the secreting stomach replacing isotonic saline with isotonic HCl as the bathing fluid on the mucosal surface, results in a change in the serosal to mucosal PD of only 19 mv, which is 40% less than the liquid junction potential between gastric juice and saline. In the surface epithelial cells of both resting and secreting stomach, multiple levels of potentials were found. For the secreting stomach, the resistance between the interstitial fluid of the pit region and the fluid on the mucosal surface was 55 ohm cm², determined as the change in PD per unit of applied current across stomach. The implications of these findings are discussed with reference to the separate site theory of HCl formation.