Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.     

Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes

Chondroitin 6-sulfotransferase, which transfers sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-acetylgalactosamine in chondroitin, was purified 1,430-fold to apparent homogeneity with a 22% yield from the serum-free culture medium of chick embryo chondrocytes by affinity chromatography on heparin-Sepharose CL-6B, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single broad protein band with an apparent molecular weight of 75,000. Since the purified enzyme has an apparent molecular weight of 160,000 as judged by gel chromatography on Superose 12, the active form of chondroitin 6-sulfotransferase may be a dimer. The purified enzyme transferred sulfate to chondroitin, chondroitin sulfate, and corneal keratan sulfate. Chondroitin sulfate E from squid cartilage, dermatan, sulfate, and heparan sulfate hardly served as acceptors of the sulfotransferase. The sulfated product derived from keratan sulfate was degraded by keratanase but not by chondroitinase ABC.     

Preparation of N epsilon B28-monoazidobenzoyl insulin-like growth factor I and photoaffinity labeling of insulin-like growth factor I receptor

Recombinant human insulin-like growth factor I (hIGF-I) was reacted with azidobenzoyl hydroxysuccinimide to produce a mixture of photoactive hIGF-I derivatives. The mixture was purified by reversed-phase HPLC to yield three mono-substituted azidobenzoyl hIGF-Is. One of the derivatives was identified by amino acid sequencing as N epsilon B28-monoazidobenzoyl hIGF-I. This derivative was indistinguishable from native hIGF-I when bioassayed in Rat-1 fibroblasts. A 120-kDa band, the alpha subunit of the IGF-I receptor, was specifically labeled in Rat-1 plasma membranes by this photoprobe. The labeling of this band was reduced by hIGF-I at 1 nM and completely abolished by hIGF-I, but not insulin, at 100 nM, indicating the specificity of the photolabeling of the IGF-I receptor by this fully active IGF-I photoprobe.     

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.     

Purification and some properties of ribonuclease from Xenopus laevis eggs

A 122 kDa RNase from eggs of Xenopus laevis was purified by sequential chromatography on Sephadex G-75, DEAE-cellulose, heparin-Sepharose and TSK gel G3000SW columns, and gave a single 60 kDa band on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The RNase composed of two 60 kDa subunits is able to recognize pyrimidine bases specifically. The pH optimum of the RNase was 7.5 in Tris-HCl buffer. The enzyme activity was abolished by treatment at 80 degrees C for 5 min and pH 2 or 12 for 1 h. Since egg lectins with RNase activity obtained from Rana catesbeiana and R. japonica and bovine pancreatic RNase A show about 30% protein homology and these three proteins are 12-14 kDa heat-stable RNases, [K. Titani, K. Takio, M. Kuwada, K. Nitta, F. Sakakibara, H. Kawauchi, G. Takayanagi and S. Hakomori, Biochemistry, 26, 2189 (1987); Y: Kamiya, F. Oyama, R. Oyama, F. Sakakibara, K. Nitta, H. Kawauchi, Y. Takayanagi and K. Titani, J. Biochem. (Tokyo), 108, 139 (1990)], the data suggest that the X. laevis egg RNase is a unique protein compared with RNases from not only amphibians, but also mammals.     

Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body (Bombyx mori)

NADH-dependent glutamate synthase (Nadh-Gogat; EC 1.41.14) was purified 766-fold from the fat body of 5th instar larvae of the silkworm with a final specific activity of 13.8 units/mg protein by a produce including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200 HR gel filtration. The purified enzyme yielded a single band corresponding to a molecular mass of 195kDa by SDS-polyacrylamide gel electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the enzyme is composed of a monomeric polypeptide. The enzyme showed an absorption spectrum with maximum values at 272, 375, and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-terminal amino acid sequence showed a high similarity to those of other GOGATs from plants, yeast and bacteria, but no similarity to other known proteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which Km values for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 micro M, respectively. Azaserine, 6-diazo-5-oxonorleucine and p-chloromercuribenzoic acid were strong inhibitors of the activity. These result show that NADH-GOGAT in the silkworm fat body strongly resembles other eukaryotic NADH-GOGATs, suggesting that it plays a significant role in ammonia assimilation in the same manner as the other enzymes.     

Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells

Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L-iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ovary cells. The isolation procedure included a combination of affinity chromatography on heparin-Sepharose CL-6B and 3',5'-ADP-agarose, which was repeated twice for each, and finally gel chromatography on Superose 12 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin and mouse Engelbreth-Holm-Swarm tumor heparan sulfate were used as acceptors, the purified enzyme transferred sulfate to position 2 of L-iduronic acid residue but did not transfer sulfate to the amino group of glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan sulfates from pig aorta and bovine liver, however, were poor acceptors. The enzyme showed no activities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme activity was around 5.5. The enzyme activity was minimally affected by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.20 microM.     

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: purification and properties in comparison with liver cathepsin D

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.     

Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity

A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.     

The tensor apparatus in double-orifice mitral valve: interpretation of echocardiographic findings

Carboxylesterases (EC 3.1.1.1) from human liver were purified using Q-Sepharose, Sephadex G-150, isoelectrofocusing and Con A-Sepharose. The calculated molecular mass of the pI 5.3 enzyme was 120 kDa and 61 kDa from the results of Sephadex G-150 gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that this enzyme is a dimer. On the other hand, carboxylesterase pI 4.5, with a molecular mass of 64 kDa, was a monomer. The activities of both enzymes were inhibited by typical serine enzyme inhibitors. Amino acid sequence analysis of the purified enzymes pI 5.3 and 4.5 showed high homology with rabbit carboxylesterase form 1 and 2, respectively. The results also suggested that carboxylesterase pI 5.3 is identical to the deduced amino acid sequence from cDNA for HU1, and that carboxylesterase pI 4.5 is identical to the deduced amino acid sequence from the cDNA registered as human carboxylesterase (hCE-2) in GenBank. We first purified carboxylesterase pI 4.5 and investigated its hydrolytic activity upon various drugs. The two enzymes differed in substrate specificity. Prodrugs of angiotensin-converting enzyme inhibitors, such as delapril and imidapril, were converted to active metabolites by carboxylesterase pI 5.3, but not by carboxylesterase pI 4.5. The hydrolysis velocity of temocapril by carboxylesterase pI 5.3 was 12-fold faster than by carboxylesterase pI 4.5. In contrast, aspirin, oxybutynin and procaine were hydrolyzed by only carboxylesterase pI 4.5. We also found that an amide-linkage in drugs, except for that in aniracetam, was not a good substrate for the two enzymes. Consequently, carboxylesterases pI 5.3 and 4.5 may be involved in the metabolism of various drugs containing an ester-linkage.     

Purification to apparent homogeneity and properties of pig kidney L-fucose kinase

L-Fucokinase was purified to apparent homogeneity from pig kidney cytosol. The molecular mass of the enzyme on a gel filtration column was 440 kDa, whereas on SDS gels a single protein band of 110 kDa was observed. This 110-kDa protein was labeled in a concentration-dependent manner by azido-[32P]ATP, and labeling was inhibited by cold ATP. The 110-kDa protein was subjected to endo-Lys-C digestion, and several peptides were sequenced. These showed very little similarity to other known protein sequences. The enzyme phosphorylated L-fucose using ATP to form beta-L-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme was D-arabinose, at about 10% the rate of L-fucose. Many of the properties of the enzyme were determined and are described in this paper. This enzyme is part of a salvage pathway for reutilization of L-fucose and is also a valuable biochemical tool to prepare activated L-fucose derivatives for fucosylation reactions.     

Successful treatment of intraoperative myocardial ischemia with nicorandil

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.     

Evidence for cell surface and internal phospholipase activity in ascidian eggs

Glutaredoxin, also known as thioltransferase, was purified from Cryptococcus neoformans by procedures including DEAE-cellulose ion exchange chromatography, Q-Sepharose ion-exchange chromatography, and gel filtration on Sephadex G-50. Its purity was confirmed by SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 12,000 Da. The purified enzyme has a K(m) value of 1.03 mM with 2-hydroxyethyl disulfide as a substrate. The enzyme also utilizes L-sulfocysteine, L-cystine, and bovine serum albumin as substrates in the presence of reduced glutathione. The enzyme has K(m) values of 0.34-2.50 mM for these substrates. It was greatly activated by thiol compounds such as reduced glutathione, dithiothreitol, L-cysteine and beta-mercaptoethanol. It is partially inactivated at 60 degrees C or higher temperatures. It plays an important role in thiol-disulfide exchange in Cryptococcus neoformans.     

Large intracranial vessel occlusive vasculopathy after radiation therapy in children: clinical features and usefulness of magnetic resonance imaging

The marine rotifer, Brachionus plicatilis, is able to digest Chlorella efficiently, suggesting that the rotifer contains a powerful cellulolytic enzyme system. A multi-component cellulolytic complex, including endoglucanase (CM-cellulase), cellobiohydrolase and beta-glucosidase, was found in Brachionus plicatilis. Endoglucanase (endo-beta-1,4 glucanase) was purified to homogeneity from rotifer homogenates using a sequential chromatographic method. The purified enzyme exhibits a strong hydrolytic activity with carboxymethyl(CM)-cellulose. The optimum temperature and pH for the endoglucanase activity were 37 degrees C and 7.0, respectively. 80% of the CM-cellulase activity was retained in salt mixture that ranged from 150 to 500 mM NaCl equivalent. The purified protein was isolated with a molecular weight of approximately 62 kDa estimated by SDS-polyacrylamide gel electrophoresis.     

Arteriosclerosis obliterans that was improved by LDL apheresis

An enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatography, gel filtration with Superdex 200, DEAE ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140,000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1 mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu- Ala-Leu .     

The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).     

Purification and characterization of the acid soluble 26-kDa polypeptide from soybean seeds

Whey proteins from soybean seeds of Japanese varieties were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Among 11 varieties of soybean, three green and one black soybeans lacked a 26-kDa band that was found in all yellow soybeans. In this paper, the 26-kDa protein was named AS26k (acid soluble 26-kDa protein) temporarily. The AS26k protein was purified from Glycine max cv. Nattosyoryu, which is yellow soybean, through four purification steps: 30-35% saturated ammonium sulfate fractionation, ion exchange chromatography on S Sepharose Fast Flow, gel filtration on Sephadex G-100, and hydrophobic chromatography on phenyl Sepharose CL-4B. Purified AS26k was cleaved with V8 proteinase from Staphylococcus aureus or CNBr. The cleaved polypeptide contained two typical dehydrin motif sequences: DEYGNPV and (M)DKIKEKLPG, and a 19 amino acids sequence similar to a pea dehydrin. Native AS26k had a molecular mass of 32 kDa on gel filtration and a pl of 7.2 on two-dimensional PAGE. Similarly to other dehydrins and late embryogenesis abundant (LEA) proteins, AS26k was rich in hydrophilic amino acids, and highly heat stable. These results showed that AS26k was a dehydrin, a group II LEA protein in soybean seeds.     

Human vitreous hyaluronidase: isolation and characterization

PURPOSE: Hyaluronic acid (HA) is the predominant glycosaminoglycan (GAG) of the human vitreous. Interaction of this HA with vitreous collagen is important for maintaining gel structure. The mechanism of HA homeostasis in the vitreous is incompletely understood. The aim of this study was to determine whether hyaluronidase, an endoglycosidase that degrades HA, was present in human vitreous. METHODS: Vitreous samples were collected from post-mortem eye bank specimens and from non-hemorrhagic, non-inflamed biopsy specimens. Vitreous hyaluronidase was purified by a series of column chromatographic steps, and its activity was measured by an ELISA-like assay and by substrate gel electrophoresis through and HA-impregnated gel. The purified hyaluronidase was also analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting. RESULTS: Hyaluronidase activity was detected in vitreous samples from both post-mortem and biopsy specimens. The enzyme was most active at acid pH, but demonstrated significant activity at neutral pH. The partially purified enzyme migrated as a 59 kDa protein on SDS-PAGE, and a single band on Western blots. CONCLUSIONS: Hyaluronidase is present in the human vitreous. Thus, hyaluronidase may be involved in HA catabolism in the vitreous and may play a role in determining its gel structure.     

Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.     

Immunological characterization, lipid dependence, and subcellular localization of CTP:phosphoethanolamine cytidylyltransferase purified from rat liver. Comparison with CTP:phosphocholine cytidylyltransferase

CTP:phosphoethanolamine cytidylyltransferase (ET) (ethanolamine-phosphate cytidylyltransferase, EC 2.7.7.14), which is generally considered as the rate-regulatory enzyme of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, was purified to homogeneity from a rat liver postmicrosomal supernatant. A polyclonal antibody was raised against the enzyme in rabbits and subsequently purified by affinity chromatography. The affinity-purified antibody recognized one single immunoreactive 49.6-kDa protein band on SDS-polyacrylamide gel. The enzyme showed an isoelectric point at a pH of 6.5 and was sensitive to various sulfhydryl reagents. Cross-reactivity experiments of ET and CTP:phosphocholine cytidylyltransferase (CT) (choline-phosphate cytidylyltransferase, EC 2.7.7.15) with their corresponding antibodies showed that these enzymes were immunologically distinct. In contrast with the well known lipid dependence of CT, the activities of both purified and cytosolic ETs were not affected by the presence of various phospholipid preparations. Differential centrifugation studies as well as release experiments with digitonin-permeabilized hepatocytes demonstrated that ET, unlike CT, is not associated with cellular organelles. However, amino acid analysis of ET revealed a high content of hydrophobic amino acids, suggesting a possible association of this enzyme with some kind of cellular structure in the hepatocyte.