The localization in rat liver of alkaline phosphodiesterase to a discrete organelle implicated in ligand internalization

The subcellular distribution of alkaline phosphodiesterase and NADH pyrophosphatase, two activities thought to be expressed by the same enzyme, was investigated. Although the two activities share a localization to a low-density vesicular membrane (equilibrium density = 1.12 g.cm-3), little NADH pyrophosphatase activity, in contrast to alkaline phosphodiesterase, was found in plasma membrane (equilibrium density = 1.18 g.cm-3), as reflected by the distribution of 5'nucleotidase. The binding and uptake of 125I-labelled insulin in perfused rat liver was also investigated. This ligand was found to bind to sinusoidal plasma membrane at 4 degrees C, but was rapidly internalized at 37 degrees C to the low-density membrane, which is rich in alkaline phosphodiesterase and NADH pyrophosphatase. These vesicular membranes were shown to belong to none of the enzymatically characterized subcellular bodies, and it is proposed that they represent discrete organelles participating in the subcellular processing of receptor-ligand complexes.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.     

Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.     

Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis.

sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.     

Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.     

The silk-producing system ofLinyphia triangularis (Araneae,Linyphiidae) and some comparisons with Araneidae

Summary The spinning apparatus ofLinyphia triangularis, adult females and males, was studied with the scanning electron microscope and the main anatomical and histochemical characteristics of the silk glands, including the epigastric apparatus of males, are presented. The epigastric glands seem to be important for the construction of sperm webs. A detailed account of the use of the different kinds of silk in web building is given.The spinning apparatus ofLinyphia closely corresponds to the araneid pattern. Characteristic of linyphiid spiders is the poor development of the aciniform glands. Corresponding to the minor importance of capture threads forLinyphia, the triads (aggregate and flagelliform glands) are less developed than in Araneidae.Linyphia make much less use of the secretions of the piriform glands for connecting threads than Araneidae. Capture threads adhere to other threads by their own glue; other threads seem mostly to be bound to one another by the secretion of the minor ampullate glands whose chemical properties, inLinyphia, appear especially adapted to this function. Neither the anatomical and histochemical data concerning the spinning apparatus nor the structure of the webs provide any indication of close relationships between Linyphiidae and Agelenidae, as was recently claimed.     

Topology of the anion-selective porin Omp32 from Comamonas acidovorans.

Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anion-selective porin Omp32. Proteinase K treatment above a critical temperature of 42 degrees C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the structural basis of the anion selectivity.     

Alteration of cell surface carbohydrates associated with ordered and disordered proliferation of oral epithelia: a lectin histochemical study in oral leukoplakias, papillomas and carcinomas

Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without (n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n = 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B4). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Gal beta 1-3GalNAc), HPA (D-GalNAc alpha) and GS I-B4 (alpha D-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia. Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/disordered proliferation.     

Indium-111 autologous leucocyte scanning: comparison with radiology for imaging the colon in inflammatory bowel disease.

Indium-111 autologous leucocyte scanning was compared with barium enema for assessing the extent of colonic disease in Crohn''s colitis and ulcerative colitis. Scanning was shown to be as accurate as conventional radiology in colitis, reliably distinguishing active from inactive disease. The results suggest that 111In-leucocyte scanning is an accurate, non-invasive, alternative technique for imaging the extent of disease in colitis.