Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Antilipolytic activity in vitro of various analogs of nicotinic acid. Structure-activity relationship

The antilipolytic activity of a series of N aryl-nicotinamides and of alpha picolinic acid, has been tested in vitro. Lipolysis was stimulated by epinephrine (20 micrograms/ml of incubation medium) using rat's epididymal adipose tissue slices. Only N(2-carboxy methyl phenyl) nicotinamide showed antilipolytic effect comparable to that of nicotinic acid at similar concentrations (2 X 10(-5) M). Picolinic acid (10(-4) M) showed no antilipolytic effect. These results, together with those of the literature, are discussed in regard to the relations between structure and antilipolytic activity.     

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.     

Possible effect of bromocriptine on the insulin receptor

The administration of 0.00011 mg/g weight/day of bromocriptine (CB154) for 7 days to Wistar rats, improved the peripheral glucose uptake without significant changes in plasma insulin level, during the intravenous glucose tolerance test (0.33 g/kg). The mode of the bromocriptine action on binding of 125I insulin to erythrocyte insulin receptors has been evaluated. The total number of sites was greater with bromocriptine (513.1 +/- 124.1 pM/1,CB154 815.6 +/- 107.9 pM/l) (p less than 0.01). The high affinity/low capacity compound of insulin receptor, in CB154 rats (51.8 +/- 16.8 pM/l) was higher than in normal rats (18.3 +/- 8.9 pM/l) (p less than 0.005). Additional studies indicated that CB154 had no effect on the rate of association and dissociation of 125I insulin from rats erythrocyte insulin receptors. The degradation of insulin or the erythrocyte receptor sites do not change, after the treatment with CB154.     

Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii

In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii. Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology. EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions. Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells.     

Presence of pH2-sensitive circulating interferon among Callithrix jacchus marmosets

Significant and relatively stable levels of serum-interferon were demonstrated in a Callithrix jacchus population. This circulating interferon was acid-sensitive in all cases, classifying it as immune or "gamma-type" interferon. Our results in these hematopoietic chimeras suggest that the presence of immune-type or at least pH 2-sensitive interferon could be related to the presence of two allogenic lymphocyte populations in each marmoset.     

Phospholipid methylation in pancreatic islets

Glucose provokes a transient stimulation of phospholipid methylation in rat pancreatic islets, possibly by increasing phospholipid methyltransferase activity. The association of DL-homocysteine and 3-deazaadenosine inhibits phospholipid methylation. The methylation of phospholipids may play a role in the stimulus-secretion coupling for glucose-induced insulin release.     

Fractionation of the gene-size macronuclear chromatin fragments of the binucleated eukaryote Oxytricha

Summary The macronuclear chromatin of Oxytrichia nova consists of chromatin fragments which are fully soluble in 0.2 mM EDTA and whose DNA length varies from 500–25 000 bp. The DNA migrates electrophoretically as a series of discrete bands, with specific genes present in only one or a few bands. The chromatin fragments are composed of nucleosomes and migrate electrophoretically in proportion to their DNA length. These results suggest schemes for the fractionation of undigested chromatin in order to enrich for specific genes, facilitating analysis of changes in chromatin structure associated with changes in gene expression.     

What's new in lysozyme research?

The present review is focused on the main achievements realized in the lysozyme research field since the meeting held in 1972 to commemorate the fiftieth anniversary of the discovery of this enzyme. Despite of extensive structural, physico-chemical, crystallographic, genetic, immunological and evolutionary studies devoted to lysozymes, their biological role is still not exactly known.     

Effect of Escherichia coli lipopolysaccharide on the glucagon and insulin binding to isolated rat hepatocytes

Summary Number and affinity constant of low affinity binding sites of insulin and glucagon to isolated hepatocytes decreased when the cells were incubated with Escherichia coli 0111:B4 lipopolysaccharide. This effect agrees with a non-specific binding of lipopolysaccharide to hepatocytes, similar to the well-recognized non-specific binding of albumin. Also, binding of different lectins to their glycoprotein receptors did not affect the [¹⁴C]lipopolysaccharide interaction with the cell membrane surface. Endotoxin depresses gluconeogenesis from lactate when the precursor was incubated with the cells for short time intervals. The longer the preincubation interval with lipopolysaccharide, the higher the inhibition of gluconeogenesis in the absence and in the presence of glucagon.The effect of endotoxin was also studied on the glucagon-induced synthesis of cyclic AMP and the glucagon binding. Levels of cyclic AMP and hormone binding decreased with increasing both endotoxin concentrations and preincubation intervals at which cells were in contact with endotoxin.