Preparation and anticoagulant activity of a low-molecular-weight sulfated chitosan

Synthesis of chitosan sulfates with low molecular weight (M_v 9000–35,000 Da) was carried out by sulfation of low molecular weight chitosan (M_v 10,000–50,000 Da). The oleum was used as sulfating agent and dimethylfornamide as medium. The chitosans were prepared by enzymatic and acidic hydrolysis of initial high molecular weight chitosan as well as by extrusion solid-state deacetylation of chitin. As was shown by FT-IR and NMR-methods and elemental analysis, the sulfation occurred at C-6 and C-3 positions and substitution degree is 1.10–1.63. The molecular weight sulfated chitosan was determined by viscometric method and the Mark–Houwink equation [η]=10⁻⁵ 4.97 M^0.77. Study of anticoagulant activity showed that chitosan sulfates with lowered molecular weight demonstrated a regular increase of anti-Xa activity like heparins.     

Molecular characterization of mouse 17β-hydroxysteroid dehydrogenase IV

17β-hydroxysteroid dehydrogenases (17β-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17β-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17β-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal β-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17β-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17β-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17β-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17β-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17β-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.     

Purification and characterization of rat liver minoxidil sulphotransferase.

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Human 17β-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization

Estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a pivotal role in the synthesis of estrogens. We overproduced human placental estrogenic 17β-HSD using a baculovirus expression system for the study of the enzyme mechanism. A cDNA encoding the entire open reading frame of human 17β-HSD was inserted into the genome of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda (Sf9) insect cells. Metabolic labeling and Western blot analysis using polyclonal antibodies raised against native human 17β-HSD indicated that a molecule with an apparent mass of 35 kDa was maximally expressed 60 h after infection. At that time interval, intracellular 17β-HSD activity reached 0.26 U/mg of protein in crude homogenate, about 70 times the level measured in human placenta. Purification of recombinant 17β-HSD was achieved by a single affinity fast liquid protein chromatography step yielding 24 mg of purified 17β-HSD protein per liter of suspension culture, with a specific activity of about 8 μmol/min/mg of protein for conversion of estradiol into estrone, at pH 9.2. In addition, the recombinant protein purified from infected Sf9 cells was assembled as a dimer with molecular mass and specific activity identical to those of the enzyme purified directly from placenta. The present data show that the baculovirus expression system can provide active 17β-HSD that is functionally identical to its natural counterpart and easy to purify in quantities suitable for its physico-chemical studies.     

Purification and characterization of genetically polymorphic deoxyribonuclease I from human kidney.

Deoxyribonuclease I (DNase I) was purified about 850,000-fold from human kidney using a rabbit anti-human urine DNase I antibody and sensitive DNase I activity assay. On SDS-PAGE, the purified kidney DNase I gave a single major band, and its molecular mass was estimated to be 38,000 Da. The activity of purified kidney DNase I was dependent on the presence of Mg2+ and Ca2+. G-Actin inhibited the activity, as did the anti-urine DNase I antibody. The properties of the kidney DNase I were the same as those of urine DNase I.     

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.     

Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha

Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha). The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha. Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa). Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Molecular cloning, expression, localisation and functional characterisation of a rabbit SULT1C2 sulfotransferase.

The importance of sulfotransferases in xenobiotic metabolism is gaining recognition. The gastrointestinal (GI) tract is a major portal of entry for many xenobiotics, yet little is known about the contribution of sulfotransferases to detoxication or bioactivation metabolism in these tissues. To this end, isolation and characterisation of sulfotransferases expressed in the stomach of rabbits was undertaken. A unique sulfotransferase cDNA (GenBank Accession No. AF026304) was isolated from a rabbit stomach cDNA library. This cDNA was 1439 base pairs (bp) long and has an open reading frame of 888 bp. On expression of the cDNA in both COS cells and E. coli, a protein molecular weight of 34 kDa was detected on SDS-PAGE. Immunoblotting using an antibody raised in goats against the bacterially expressed protein detected expression of the protein in GI tract tissues. The 34 kDa immunoreactive band was detected in rabbit GI tract tissues (stomach, duodenum, jejunum, ileum, colon, caecum and rectum), liver and kidneys, but not in the lungs (n = 3). The human ortholog (GenBank Accession No AF026303) of the rabbit enzyme was cloned from a human stomach cDNA library. These two enzymes share 84% amino acid sequence identity and have been termed 1C2 sulfotransferases. When functional and kinetic characterisation of the recombinant rabbit and human proteins was carried out using 16 known ST substrates, detectable sulfonation activity was observed only with p-nitrophenol (with Km values of 2.2 mM and 13.3 mM, respectively). In conclusion, we have identified a rabbit GI tract sulfotransferase belonging to a newly defined sulfotransferase subfamily.     

The presence of glycogen synthase in phosphorylase kinase preparations isolated from rabbit skeletal muscles

Phosphorylase kinase isolated from rabbit skeletal muscle contains a protein whose molecular mass as determined by polyacrylamide gel electrophoresis is 571 000 Da. The protein was found to possess a higher affinity for glycogen as compared to phosphorylase kinase and phosphorylase. The protein separated from kinase by chromatography on a DEAE-cellulose column produced during SDS electrophoresis one protein band corresponding to Mr of 95 200 Da. The above properties of the protein and the glycogen synthetase activity revealed in the presence of glucose-6-phosphate suggest that phosphorylase kinase preparations contain a hexameric form of glycogen synthetase.     

Isolation of a cDNA clone encoding a high molecular weight precursor to a 6-kDa pulmonary surfactant-associated protein

Mammalian surfactant is an incompletely defined mixture of lipids and associated proteins of molecular mass 35,000 Da and approximately 6,000 Da. Surfactant preparations which are highly effective in treating respiratory distress syndrome in premature infants lack the 35-kDa proteins, but contain the 6-kDa proteins. We isolated and partially sequenced one of these low molecular weight proteins from the lung lavage material of an alveolar proteinosis patient. Oligonucleotides deduced from the sequence were used as probes to isolate a human cDNA clone. The clone codes for a 42-kDa protein which contains the sequence of the 6-kDa protein. Messenger RNA coding for the 42-kDa protein was identified in human lung RNA by in vitro translation and immunoprecipitation of the translation products with an antiserum against purified bovine surfactant 6-kDa proteins. Immunoprecipitation of the 42-kDa primary translation product is inhibited by the presence of the bovine 6-kDa protein. These observations suggest a precursor-product relationship of the 42-kDa protein to one of the 6-kDa proteins.     

The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.     

Complete nucleotide sequence of band 3 related anion transport protein AE2 from human kidney.

The cDNA coding for the complete human band 3 related anion exchange protein AE2 has been cloned from human kidney mRNA. The protein is encoded by a mRNA of approx. 3885 nucleotides containing an open reading frame of 3720 nucleotides. The AE2 protein consists of 1240 amino acid residues and has a molecular mass of 136,805 Da.     

The ARF-like 2 (ARL2)-binding protein, BART. Purification, cloning, and initial characterization.

ARF-like proteins (ARLs) comprise a functionally distinct group of incompletely characterized members in the ARF family of RAS-related GTPases. We took advantage of the GTP binding characteristics of human ARL2 to develop a specific, high affinity binding assay that allowed the purification of a novel ARL2-binding protein. A 19-kDa protein (BART, Binder of Arl Two) was identified and purified from bovine brain homogenate. BART binding is specific to ARL2.GTP with high affinity but does not interact with ARL2.GDP or activated ARF or RHO proteins. Based on peptide sequences of purified bovine BART, the human cDNA sequence was determined. The 489-base pair BART open reading frame encodes a novel 163-amino acid protein with a predicted molecular mass of 18,822 Da. Recombinant BART was found to bind ARL2.GTP in a manner indistinguishable from native BART. Northern and Western analyses indicated BART is expressed in all tissues sampled. The lack of detectable membrane association of ARL2 or BART upon activation of ARL2 is suggestive of actions quite distinct from those of the ARFs. The lack of ARL2 GTPase-activating protein activity in BART led us to conclude that the specific interaction with ARL2.GTP is most consistent with BART being the first identified ARL2-specific effector.     

Cloning and expression of Taxus acyltransferase cDNA

A new full-length acyltransferase cDNA was obtained from Taxus chinensis by homology-based cloning strategy. The cDNA has an open-reading frame of 1,275 nucleotides, which encodes 425 amino acids with a calculated molecular weight of 47,241 Da and an estimated pI value of 5.93. The deduced amino acid sequence resembles the sequences of other cloned acyltransferases (56-61% identity; 71-75% similarity) involved directly in taxol biosynthetic pathways. This cDNA was expressed in Escherichia coli using the expression vector pET32a(+). The expression band corresponds to the calculated mass plus the N-terminal fusion protein derived from the vector.     

Expression and characterization of the human 3β-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)

The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3β-hydroxysteroids, 3-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens. In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3β-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol. There was no activity detected towards testosterone, dexamethasone, β-estradiol, androsterone, or p-nitrophenol. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals. Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta. No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue. Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody. The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis. The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.     

Cloning and characterization of the annexin II receptor on human marrow stromal cells

Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.