(1/2317) Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.
A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 126.96.36.199) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family. (+info)
(2/2317) Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning.
Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid. (+info)
(3/2317) The relationship of glycosylation and isoelectric point with tumor accumulation of avidin.
Radiolabeled avidin markedly accumulated in intraperitoneal tumors and was cleared rapidly from circulation when given intraperitoneally. This study investigated the mechanisms of the tumor localization of avidin. METHODS: Avidin was deglycosylated through endoglycosydase-H digestion and/or neutralized by acetylation of its lysine amino acids with acetic acid N-hydroxysuccinimide ester. Avidin and modified avidins were analyzed using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and isoelectric focusing. A tumor model was established by intraperitoneal injection of human colon cancer cells, LS180, in nude mice. Avidin and modified avidins were labeled with 111In using diethyleneamine pentaacetic acid-biotin and were administered intraperitoneally into the tumor-bearing mice. The biodistribution of radioactivity was examined 2 and 24 h postinjection. RESULTS: Deglycosylated avidins revealed a major band of smaller molecules on SDS/PAGE. The isoelectric point of neutralized avidins was reduced to less than 5, whereas that of unneutralized avidins was more than 9.5. Biodistribution study demonstrated that liver uptake was decreased by deglycosylation and kidney accumulation was decreased by neutralization, respectively. The blood clearance was remarkably slowed by combined modification of deglycosylation and neutralization. The tumor uptake of radioactivity was reduced by either deglycosylation or neutralization and was further decreased with combined modification. CONCLUSION: Both high glycosylation and positive charge of avidin contributed to its accumulation in tumor. This study may facilitate development of a new vehicle for the delivery of therapeutic agents to intraperitoneal tumors. (+info)
(4/2317) The purification and characterization of rabbit placental lactogen.
Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts. (+info)
(5/2317) Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase.
Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m). NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione. (+info)
(6/2317) Rapid purification of membrane extrinsic F1-domain of chloroplast ATP synthase in monodisperse form suitable for 3D-crystallization.
A new chromatographic procedure for purification of the membrane extrinsic F1-domain of chloroplast ATP synthase is presented. The purification is achieved by a single anion exchange chromatography step. Determination of the enzyme-bound nucleotides reveals only 1 mole of ADP per complex. The purified enzyme shows a latent Ca(2+)-dependent ATPase activity of 1.0 mumol.mg-1 min-1 and a Mg(2+)-dependent activity of 4.4 mumol.mg-1 .min-1. Both activities are increased up to 8-10-fold after dithiothreitol activation. Analysis of the purified F1-complex by SDS/PAGE, silver staining and immunoblotting revealed that the preparation is uncontaminated by fragmented subunits or ribulose-1,5-bisphosphate carboxylase/oxygenase. Gel filtration experiments indicate that the preparation is homogenous and monodisperse. In order to determine the solubility minimum of the purified F1-complex the isoelectric point of the preparation was calculated from pH mapping on ion exchange columns. In agreement with calculations based on the amino acid sequence, a slightly acidic pI of 5.7 was found. Using ammonium sulphate as a precipitant the purified CF1-complex could be crystallized by MicroBatch. (+info)
(7/2317) Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via alpha2beta1 integrin.
Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35 degrees C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin alpha2beta1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4 h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30 degrees C. (+info)
(8/2317) Calcium-binding protein S100A7 and epidermal-type fatty acid-binding protein are associated in the cytosol of human keratinocytes.
Expression of epidermal-type fatty acid-binding protein (E-FABP) and S100A7 has previously been shown to be elevated in psoriatic skin, a disease characterized by abnormal keratinocyte differentiation. However, no causal relationship between the up-regulation of these proteins and the disease has been shown. E-FABP is thought to be involved in cytosolic fatty acid (FA) transport, whereas the role of S100A7 is still unknown. In this report, we show by overlay assays that E-FABP, immobilized on nitrocellulose, is able to capture S100A7 from cytosolic psoriatic protein extracts and vice versa, suggesting the formation of a complex between the two proteins. Using purified E-FABP and S100A7, the complex can be reconstituted only in presence of EDTA. Moreover, we show that increased EDTA concentrations in psoriatic cytosolic protein extracts enhance complex formation. Partial complex disruption was obtained by the addition of physiological concentrations of Zn2+ (0.1 mM), whereas Ca2+ at 5 mM and Mg2+ at 30 mM had no effect. On the other hand, high Ca2+ concentrations (30 mM) resulted in partial complex disruption. Oleic acid-binding properties were observed for free E-FABP and the complex E-FABP-S100A7, but not for free S100A7. By using confocal microscopy we show that S100A7 and E-FABP are co-localized in the cytoplasm of differentiating keratinocytes from lesional psoriatic skin. These data indicate that formation of the E-FABP-S100A7 complex and its FA-binding function might be regulated at least by bivalent cations. (+info)