Ribulose bisphosphate-induced, slow conformational changes of spinach ribulose bisphosphate carboxylase cause the two types of inflections in the course of its carboxylase reaction.

The cause of the inflection in the course of the carboxylase reaction and the changes in the functioning form of spinach ribulose bisphosphate carboxylase (RuBisCO) during the reaction were elucidated by relating the activity to the protein conformation of RuBisCO using a fluorescence probe, 2-p-toluidinylnaphthalene sulfonate. The activity of RuBisCO in the linear phase was 50 to 60% of that in the initial burst at 0.5 to 1.0 mM ribulose bisphosphate (RuBP) and 65 to 80% at 2 to 5 mM RuBP. The amount and the progress of the decrease in the activity during the reaction had a close relationship to a change in the protein conformation of RuBisCO. The enzyme, the substrate binding sites of which were masked beforehand with carboxyarabinitol bisphosphate, still showed a change of its protein conformation upon addition of RuBP, suggesting that RuBisCO has two (substrate and regulatory) RuBP-binding sites per RuBisCO promoter. RuBisCO required over 2 mM RuBP for binding on the regulatory sites. Both sites also bound 6-phosphogluconate. When both sites were masked with 6-phosphogluconate beforehand, the course of the subsequent carboxylase reaction was linear with time. From these results, I propose that the inflection in the course of the reaction of spinach RuBisCO is a hysteretic response of the enzyme to RuBP bound to both substrate and regulatory sites.     

Differential effects of phospholipids on two similar forms of UDP-glucuronyltransferase purified from rat liver and kidney microsomes

Two isoforms of UDP-glucuronyltransferase purified from rat liver (named GT-1) and kidney (named GT-2) have various properties in common but differ in their NH2-terminal sequences. In this study, the two forms were further found to have common immunochemical properties, i.e., they could not be distinguished by Ouchterlony double diffusion and immunoblotting analyses. These isoforms also had the same inducibility as shown by immunoblotting analysis: GT-2 protein in rat was increased by treatment with beta-naphthoflavone and 3-methylcholanthrene, whereas GT-1 was inducible by 3-methylcholanthrene. However, the effects of phospholipids on these enzymes were extremely different. 1-Naphthol glucuronizing activity of GT-1 was increased 7.5-8-fold by lysophosphatidylcholine, but the activity of GT-2 was increased only 3-3.6-fold. The transferase activity of GT-1 toward 4-methylumbelliferone was increased 2-2.5-fold by dilauroylphosphatidylcholine, but that of GT-2 was reduced, while its 4-nitrophenol glucuronidation activity was increased 1.5-fold by the phospholipid. These results indicate that the two similar UDP-glucuronyltransferases from rat liver and kidney interact differently with phospholipids and that the activation level of UDP-glucuronyltransferase activity with phospholipids depends on the aglycone substrates.     

Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum.

The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsP3 receptor protein is composed of four noncovalently bound identical subunits each with a Mr of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2(+)-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [alpha-32P]8-azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the Bmax and Kd values for ATP binding of 2.3 pmol/micrograms and 17 microM, respectively. Reconstitution of the purified InsP3 receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 mM Ca2+) and sodium conductance (21 pS in 100-500 mM asymmetric Na+ solutions) with permeability ratios of PCa/PTris = 6.3 and PNa/PCl = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.     

Increased secretion of brain natriuretic peptide and atrial natriuretic peptide, but not sufficient to induce natriuresis in rats with nephrotic syndrome.

The levels of immunoreactive brain natriuretic peptide (ir-BNP) and immunoreactive atrial natriuretic peptide (ir-ANP) were evaluated by radioimmunoassay in both the atrium, ventricle and plasma of adriamycin-induced nephrotic rats and control rats. There was no difference in right and left atrial concentrations of ir-BNP, however, a higher right atrial concentration of ir-ANP was observed in nephrotic rats than in controls (p less than 0.01). The ventricular ir-BNP and ir-ANP were increased in nephrotic rats compared to controls (BNP: p less than 0.001, ANP: p less than 0.001). Cardiac BNPs were composed of pro-BNP (gamma-BNP) and its C-terminal 45-amino-acid peptide (BNP-45). The ratio of BNP-45/gamma-BNP in nephrotic rats was higher than that of controls in both atria and in the ventricle. Plasma ir-BNP and ir-ANP were significantly higher in nephrotic rats than in controls (BNP: p less than 0.001, ANP: p less than 0.001), and each level was negatively correlated with urinary sodium excretion in nephrotic rats (BNP: r = -0.84, p less than 0.001, ANP: r = -0.88, p less than 0.001). These results suggest that production and secretion of both BNP and ANP are concomitantly stimulated by a decreased renal ability to eliminate sodium and water, but this secretion is insufficient to induce effective natriuresis in nephrotic rats.     

Choline deficiency activates phospholipases A2 and C in rat liver without affecting the activity of protein kinase C

There is evidence to suggest that liver tumor promoters exert their effect through the interference of signal transduction in hepatic cells. Both phospholipase A(2) and phospholipase C play important roles in the generation of second messengers and in the activation of Ca(2+), phospholipid-dependent protein kinase C. Using male Sprague-Dawley rats, we investigated whether liver tumor-promoting regimens of a choline-deficient diet and phenobarbital alter the activities of phospholipase A(2) and phospholipase C in the liver, and extended the study to determine the effect of a choline-deficient diet on protein kinase C activities. Feeding a choline-deficient diet for 1 week increased the activities of both phospholipase A(2) (50%) and phospholipase C (22%), and the activities of both enzymes were more than doubled after 4 weeks. Feeding a phenobarbital diet resulted in a slight decrease in phospholipase A(2) activities at 4 weeks but no significant changes in PLC activities. The protein kinase C activities and its distribution between soluble and particulate fractions remained unchanged after 1, 2, and 4 weeks feeding of a choline-deficient diet. Thus, activation of both phospholipase A(2) and C is distinct for a choline-deficient diet, not shared by phenobarbital diet. Increased activities of these enzymes were not associated with the activation of protein kinase C under the present experimental condition.     

Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xanthine excretion in a desert scorpion,Paruroctonus mesaensis

Summary The nature of the nitrogen excretory products of a desert scorpion (Paruroctonus mesaensis) was investigated. Two dimensional thin-layer chromatography revealed one major component, comigrating with xanthine, and a minor component which occurred sporadically and comigrated with hypoxanthine. The identity of xanthine as the major nitrogenous waste was confirmed with additional chromatographic systems, by ultraviolet spectrophotometry, and by enzymatic analysis. P. measensis excretes over 90% of its waste nitrogen in the form of xanthine, and is the first animal shown to be primarily xanthotelic. Most other arachnids excrete primarily guanine. No guanine or uric acid was detected inP. mesaensis urine, although some uric acid, probably of dietary origin, was found in fecal material. Some possible mechanisms for, and consequences of, xanthine excretion are discussed.