Rabbit β-glucuronidase. Purification and properties, and the existence of multiple forms

1. beta-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH(4))(2)SO(4) fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The K(m) value for p-nitrophenyl beta-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg(2+) ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5-5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.     

Studies on glucarate catabolism: the oxodeoxyglucarate aldolase activity of glucarate hydrolyase from Pseudomonas acidovorans (Short Communication)

Glucarate hydro-lyase was isolated and purified to near homogeneity from cells of Pseudomonas acidovorans grown on glucarate. By using gel filtration and ion-exchange chromatography, it was shown that the oxodeoxyglucarate aldolase activity observed in such extracts is associated with the glucarate hydro-lyase protein.     

Generic Identification of L-Forms by Polyacrylamide Gel Electrophoretic Comparison of Extracts from Parent Strains and Their Derived L-Forms

A polyacrylamide gel electrophoretic method was used for identification of L-forms in the genera Streptococcus, Staphylococcus, and Proteus, by comparing phenol-acetic acid-water extracts of homologous parent L-form pairs to one another and to other pairs. The method requires only milligram quantities of material for analysis. The standard patterns for the strains used in this study are shown as pictures of the gels and as densitometric tracings of appropriate gels. They show that, despite occasional minor differences in some organisms, the gel electrophoretic patterns of homologous L-forms and bacterial pairs are sufficiently similar-as well as sufficiently dissimilar from patterns of other genera-to permit generic identification of an unknown L-form by reference to patterns derived from possible parental bacteria.     

Secretory IgA in Urinary Tract Infections

Secretory IgA, measured by radial immunodiffusion, was compared in the urine of children with chronic and recurrent non-obstructive urinary tract infections with that in normal children. IgA, IgG, and IgM were also measured. Absent and low levels of IgA(s) were found in both groups; however, the mean levels of IgA(s) were significantly higher in the infected group compared with normals—3·3 to 0·78 mg./24 hours, respectively. Secretory IgA was found to be locally produced in the bladder. It is suggested that IgA(s) levels reflect an antibody response to infection.     

Genetic Integration in the Heterospecific Transformation of Haemophilus influenzae Cells by Haemophilus parainfluenzae Deoxyribonucleic Acid

The in vivo chemical linkage of Haemophilus parainfluenzae deoxyribonucleic acid (DNA) with the H. influenzae genome has been found to occur at a much higher level than is suggested by the low efficiency of the heterospecific transformation of an antibiotic resistance marker. This linkage, about 60% of the level with homospecific DNA, was found to involve alkali-stable bonding. The amount of host DNA label released (about 60%) was about the same as that released during homospecific transformation. Also, over 60% of the H. influenzae cells adsorbing H. parainfluenzae DNA could not form colonies upon plating. This lethality of the heterospecific transformation was not immediate but followed considerable metabolic activity of the host cells. These data are presented to show that the "limited-pairing" hypothesis may be only a partial explanation for the low efficiency of heterospecific transformation. Another hypothesis is presented which takes into account the lethal effect of this kind of transformation.     

The `neurotoxicity'' of l-2,4-diaminobutyric acid

The neurolathyrogen l-2,4-diaminobutyric acid is concentrated by liver, and liver damage can yield neurotoxicity; thus the neurotoxicity caused by this compound may be due to liver damage followed by secondary brain damage. 1. The intraperitoneal administration of toxic doses of l-2,4-diaminobutyric acid to rats resulted in hyperirritability, tremors and convulsions in 12-20hr. and increased the concentration of ammonia of blood and brain slightly and the concentration of glutamine of brain two- to three-fold. By contrast, toxic doses of l-homoarginine, l-lysine, l-leucine and ammonium acetate caused dyspnoea, extreme prostration, and in some cases coma in 15-30min., and increased the concentration of ammonia of blood significantly and the concentration of glutamine of brain slightly. These results indicate that l-2,4-diaminobutyric acid caused a chronic ammonia toxicity, whereas the other amino acids and ammonium acetate resulted in an acute ammonia toxicity. 2. Liver slices from l-2,4-diaminobutyric acid-treated animals and normal liver slices preincubated with l-2,4-diaminobutyric acid utilized ammonia and formed urea at a lower rate than control slices from normal rats. 3. l-2,4-Diaminobutyric acid inhibited competitively ornithine carbamoyltransferase of rat liver homogenates, thus demonstrating that this reaction is a primary site of toxicity for this neurolathyrogen. Although we were unable to show marked elevations of blood ammonia concentration after treatment with l-2,4-diaminobutyric acid, these results are interpreted to mean that ammonia utilization (urea synthesis) in liver is inhibited by l-2,4-diaminobutyric acid and that at least part of the neurotoxicity is due to a prolonged slight increase in body ammonia concentration.     

Utilization of 5-Bromouracil by Thymineless Bacteria

Several thymineless Escherichia coli strains have been examined for their ability to replicate their deoxyribonucleic acid when bromouracil is substituted for thymine. The procedure we describe was used to identify a thymineless strain with characteristics relatively favorable to its use in bromouracil labeling experiments. In addition, mutants with an “absolute” thymine requirement could be easily distinguished from one with a “leaky” thymine requirement.     

The Subcellular Origin of Bioluminescence in Noctiluca miliaris

The light emitted by Noctiluca has its origin in 1 to 5 x 10⁴ organelles ("microsources") which are scattered throughout the perivacuolar cytoplasm, and which appear to be the elementary functional units of bioluminescence. Microscopical techniques, image intensification, and microphotometry were employed in their investigation. Microsources are fluorescent, strongly phase-retarding, and range widely in diameter below 1.5 microns. The number of quanta emitted in a flash from a microsource ("microflash") is of the order of 10⁵ photons. However, microflashes show a wide range of intensities, which are correlated with the size of the organelles from which they arise. Each organelle responds repetitively and with reproducible time course to a succession of invading triggering potentials. Reversible changes in the intensity of the flash emitted by the whole cell ("macroflash") occur because of graduations in intensity of microflashes rather than as a result of changes in the number of responsive organelles. The shape of the flash emitted by individual microsources resembles that of the macroflash except for slightly shorter rise and decay times. It is concluded that the macroflash results from somewhat asynchronous, but otherwise parallel summation of microflashes.     

Diabetic Neuroarthropathy: Report of Four Cases

Diabetic neuroarthropathy was observed in four patients; these are the first cases of this nature reported in the Canadian medical literature. The criteria for this diagnosis included: (1) long-standing diabetes; (2) arthropathy, most frequently involving the foot, which shows deformity, shortening and ulceration without evidence of infection or peripheral circulatory failure; (3) abolition or diminution of pain on weight-bearing; (4) diabetic peripheral neuropathy with impaired sense of position or vibration and weak or absent deep tendon reflexes. Radiographic findings were similar to those in patients with Charcot''s arthropathy from any cause.Tabes dorsalis, leprosy, syringomyelia, myelodysplasia and the arthropathies of corticosteroid therapy were ruled out in these cases. In addition to conventional medical therapy the patients were treated by means of walking-casts for several months.Diabetic neuroarthropathy is probably more common than the medical literature would indicate. Diminished sensation in the lower limbs in diabetics of long standing appears to be the major factor contributing to this disorder.