A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins

Screening for leptomycin B (LMB)-resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB-resistant mutant of S. pombe and with multicopy plasmid pDB248' as the vector led to the isolation of a gene, named pmd1+, encoding a 1362-amino-acid protein. This protein showed great similarity in amino acid sequence to the mammalian P-glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a-factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP-binding domains, as observed with the P-glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.     

Glycosaminoglycan polysulfate-induced stimulation of hyaluronic acid synthesis in rabbit knee synovial membrane: involvement of binding protein and calcium ion

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146-153]. In the present study, the interaction of [3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P less than 0.05-P less than 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.     

Amino acid sequence homology applied to the prediction of protein secondary structures, and joint prediction with existing methods

The assumption that homologous segments in different proteins may share a similar conformation is applied to the prediction of secondary structures in proteins. Sequences homologous to a target protein are searched, without allowing any gap, and compared against a number of reference proteins of known three-dimensional structure, and then a conformational state (alpha, beta or coil) for each residue of the protein is predicted by looking at the secondary structure of corresponding homologous segments. This prediction is done in a statistical rather than 'deterministic' way, by assigning the most probable conformation state among homologous data to each residue site of a target protein. A test application for 22 sample proteins yields 60% correctness on the average, a better value in comparison with two other existing methods. Joint prediction combining three methods into one is shown to increase the reliability up to 70%, when only the regions identically predicted with the three methods are taken into account. Application of the present method to 10 proteins of unknown structure is demonstrated.     

Characterization of dehydropeptidase I in the rat lung

The activity of dehydropeptidase I in rat tissues decreases in the order of lung greater than kidney greater than liver-spleen greater than other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues. Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE-cellulose column chromatography, affinity chromatography on concanavalin-A-Sepharose and high-performance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150,000 by gel filtration, comprising a homodimer of two 80,000-Mr subunits. The enzyme activity was inhibited by cilastatin, o-phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and N-ethylmaleimide-S-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to E4. Furthermore it catalyzed a hydrolytic splitting of L-Leu-L-Leu, but not S-benzyl-L-cysteine p-nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.     

Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.     

Analysis of guanase by agarose gel electrophoresis and activity staining

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.