Immunochemical and histochemical studies on a phosphonoglycosphingolipid, SGL-II, isolated from the sea gastropod Aplysia kurodai

Antiserum was raised against 3-O-MeGal beta 1----3GalNAc alpha 1----3[6' -O-(2-aminoethylphosphonyl)Gal alpha 1----2 (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Ceramide (SGL-II) isolated from the skin of a mollusc, Aplysia kurodai. This antiserum reacted with SGL-II and other phosphonoglycosphingolipids of Aplysia such as SGL-I', F-21, and some minor glycolipids on TLC plates, but it did not react with ganglioside or globoside. The sugars recognized were 3-O-methylgalactose at the non-reducing end and galactose at the branched chain of the glycolipids. One membrane glycoprotein (Mr 280,000) reacted strongly, and some other proteins reacted weakly with the antiserum. Immunohistochemical examination of the nervous tissues revealed distinct staining in the periganglionic tissue of the ganglia, and the perineural sheath of the proximal portion of the peripheral nerves. The neuropil and satellite cells were also stained. In the skin, subcutaneous connective tissues were moderately stained, and the cytoplasm of small mononuclear cells and foamy cells was also stained. The staining patterns were essentially the same in paraffin and cryostat sections. From the findings with sections pretreated with chloroform-methanol (2 : 1, v/v), it was suggested that the periganglionic and perineural stainings were due to glycoproteins, including an SDS-soluble glycoprotein of Mr 280,000, while those of the other regions were due to SGL-II and glycolipids immunologically related to SGL-II. The stainings in the skin sections were largely due to glycoproteins.     

Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.     

Synthesis of sex-specific forms of cytochrome P-450 in rat liver is transiently suppressed by hepatic monooxygenase inducers

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), and alpha-naphthoflavone (alpha-NF) on the synthesis of drug-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), and sex-specific forms of cytochrome P-450, P-450(M-1), and P-450(F-1), in male and female rats were studied. Whereas P-450(PB-1) and P-450(MC-1) in liver microsomes were markedly induced in both sexes by treatment with PB and MC, respectively, the contents of P-450(M-1) and P-450(F-1) were significantly decreased by the treatments. alpha-NF, which is not a P-450 inducer, did not change the contents of sex-specific forms of cytochrome P-450. The translatable mRNAs of the P-450s were also determined by using an in vitro translation system. The mRNAs coding for P-450(PB-1) and P-450(MC-1) were increased by drug administrations. On the other hand, the mRNAs coding for P-450(M-1) and P-450(F-1) were transiently decreased by the drugs, and then returned to the normal levels. The time courses of the induction of the drug-inducible P-450s and the repression of the sex-specific P-450s showed no close correlation. alpha-NF had no effect on the synthesis of P-450(M-1) and P-450(F-1). We also found that the synthesis of P-450(M-1) in the livers of untreated rats showed no diurnal variations.     

In vitro loss of hydrophobicity of trehalase from the brush border membrane of rabbit kidney cortex

Trehalase solubilized with 0.5% Triton X-100 and 0.5% deoxycholate from the brush border membrane of rabbit kidney cortex was all adsorbed on phenyl-Sepharose equilibrated with elution buffer containing no detergents, and all the adsorbed enzyme was eluted in one peak on the addition of 0.5% Triton X-100 to the elution buffer, in contrast to the results reported by Nakano and Sacktor (J. Biochem. 97, 1329-1335 (1985], who separated two forms of trehalase differing in hydrophobicity from rabbit kidney. On concentration of detergent-solubilized extracts, followed by incubation at 37 degrees C, however, there appeared trehalase nonadsorbable on phenyl-Sepharose, i.e. a hydrophilic trehalase. Various protease inhibitors added to the concentrated extracts did not inhibit this conversion at all. The concentration-incubation treatment also increased the proportion of trehalase that interacts with Con A-Sepharose. These results indicate that kidney trehalase that interacts with Con A-Sepharose. These results indicate that kidney trehalase is susceptible to some lytic action of a factor(s) intrinsic to the brush border membrane (limited autolysis), as seen with rabbit intestinal trehalase (Yokota et al., (1986) Biochim. Biophys. Acta 881, 405-414). Therefore, in studies of the molecular form of trehalase (and other proteins) in the brush border membrane of the kidney and intestine where a lot of hydrolases exist, it is very important to take account of limited autolysis which results in some chemical modifications without affecting enzymatic activity.     

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 microM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematoporphyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hematoporphyrin bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I]albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 micrograms/ml of albumin. The binding of hematoporphyrin-[125I]albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37 degrees C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematoporphyrin.     

Crystallization and preliminary X-ray studies on human epidermal growth factor

Single crystals of human epidermal growth factor have been prepared from a polyethylene glycol solution and characterized by X-ray diffraction. The crystals grow in a space group of P2(1) with unit cell dimensions of a = 32.7, b = 32.5, c = 22.3 A, and beta = 96.9 degrees. They contain a single molecule per asymmetric unit and show better diffraction than 2.5 A.     

Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells

The transport of 1,5-anhydro-D-glucitol (AG) across plasma membranes was investigated in rat hepatoma cells, Reuber H-35. The AG uptake by the cells showed a concentration gradient dependency: the uptake was saturated within 40 s, which was less than one-third of the saturation time for 2-deoxy-D-glucose (DG) uptake. Furthermore, the Km value of the transport system for AG was higher than 100 mM. Though AG has a pyranoid structure resembling that of glucose, AG did not compete for cellular uptake with DG, D-glucose or 3-O-methyl-D-glucose, which are taken into cells through the glucose transporters. Conversely, the DG transport was not inhibited by AG at concentrations up to 50 mM. AG transport was hardly inhibited by 10 microM cytochalasin B, which strongly inhibits glucose transporters. In contrast, the AG transport was inhibited by 100 microM phloretin much more strongly than the DG transport when cells were preincubated with the inhibitor; the inhibition constant was 28.0 microM. The AG transport was not inhibited by 100 microM phloridzin, while the DG uptake was slightly inhibited by phloridzin. On the basis of these observations we propose that the AG uptake into rat hepatoma cells is mediated by a carrier distinct from glucose transporters.     

A novel anthraquinone ring cleavage enzyme from Aspergillus terreus

An enzyme activity which catalyzes the ring cleavage of the anthraquinone questin to form benzophenone desmethylsulochrin was found in the cell-free extract of Aspergillus terreus, a (+)-geodin producer. The product was identified as desmethylsulochrin by high-resolution mass spectroscopy and chemical carrier dilution analysis. The enzyme showed an absolute requirement of NADPH and molecular oxygen. Therefore, the enzyme, named questin oxygenase, was considered to be classified as a monooxygenase. The optimum pH was around 7.5. The enzyme was very unstable and lost its activity completely after storage overnight at 4 degrees C in 0.05 M phosphate buffer, pH 7.5. The instability of the questin oxygenase was partially overcome by the addition of polyols and the non-ionic detergent Tween 80 to the buffer. By DEAE-cellulose column chromatography, two protein fractions, named DE-I and DE-II, were obtained. Neither fraction reacted with questin by itself. However, the combination of DE-I and DE-II reconstituted the questin oxygenase system to convert questin to desmethylsulochrin. This result suggested that the system is not a simple combination of oxygenase and hydrolase, but requires some additional factor(s) such as electron transfer protein.     

Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.