Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.     

The surgical treatment of necrobiosis lipoidica diabeticorum.

We review the literature on the surgical treatment of necrobiosis lipoidica diabeticorum, and we describe 7 cases treated at Stanford University Medical Center. Experiences with them prompt us to recommend surgical excision of the lesions down to the deep fascia, ligation of the associated perforating blood vessels, and the use of split-skin grafts to cover the defects. There were no recurrences when we did all these things.     

Regulation of glutaminase B in Escherichia coli. I. Purification, properties, and cold lability.

Escherichia coli contains two glutaminases, A and B, with pH optima below pH 5 and above pH 7, respectively. Neither glutaminase A nor B is released from E. coli by osmotic shock. Glutaminase B has been purified 6,000-fold and the purified preparation is estimated to contain about 40% glutaminase B. The enzyme has a molecular weight of 90,000 and an isoelectric point of 5.4. Glutaminase B exhibits a broad pH optimum between 7.1 and 9.0. Only L-glutamine is deamidated by glutaminase B, L-asparagine and D-glutamine are not deamidated. The substrate saturation curve for glutaminase B shows an intermediary plateau region. Like many regulatory enzymes, glutaminase B is cold-labile. The enzyme is inactivated by cooling and activated by warming; both processes are first order with respect to time. The activation energy for activation by warming was calculated to be 5900 cal/mol. Activation by warming increased the Vmax and decreased the S0.5 for L-glutamine, but did not alter the molecular weight of the catalytically active enzyme. Borate and glutamate protected glutaminase B from inactivation by cold.     

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.     

A histological and morphometric study on the tissue and cellular distribution of iron in carp Cyprinus carpio L. during chronic waterborne exposure

The effect of long-term (77 day) exposure of carp Cyprinus carpio to low concentrations of waterborne iron (1 mg Fe-dextran l⁻¹) on accumulation and cellular distribution of iron in the liver was assessed using Perl's staining and histological observations and morphometric measurements. Accumulation of iron in the liver occurred after 14 days of exposure, when the majority of the iron was found in the sinusoidal endothelium with lower amounts in the cytoplasm and nuclei of hepatocytes. Upon further treatment the iron was predominantly distributed in the cytoplasm of the hepatocytes either as granules or diffusive iron and in macrophages.     

Hfr formation by I pilus-determining plasmids in Escherichia coli K-12.

R483, an atypical, I pilus-determining plasmid, and also R144, a typical one, were shown to suppress the DnaA phenotype by integration into the Escherichia coli chromosome.