Sequence of Escherichia coli O128 antigen biosynthesis cluster and functional identification of an alpha-1,2-fucosyltransferase

O128 is one of the most common atypical enteropathogenic Escherichia coli isolated from diarrhea patients worldwide. The primary structure of E. coli O128 repeat units has previously been determined as -->3)-beta-D-GalNAc-(1-->4)-alpha-D-Gal-(1-->3)-beta-D-GalNAc-(1-->6)-[alpha-L-Fuc-(1-->2)]-beta-D-Gal-(1--> pentasaccharide. Here we report the complete sequencing of E. coli O128 antigen biosynthesis gene cluster and its flanking regions. Comparative sequence analysis revealed the expected O128 antigen process genes, GDP-fucose biosynthesis genes and four potential glycosyltransferase genes responsible for the assembly of E. coli O128 antigen repeats. WbsJ was shown to encode an alpha-1,2-fucosyltransferase by enzymatic assays and nuclear magnetic resonance spectroscopy analysis.     

The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis

The bacterium Streptomyces davaonensis produces the antibiotic roseoflavin, which is a riboflavin (vitamin B₂) analog. The key enzyme of roseoflavin biosynthesis is the 8-demethyl-8-amino-riboflavin-5ʹ-phosphate (AFP) synthase RosB which synthesizes AFP from riboflavin-5ʹ-phosphate. AFP is not a substrate for the last enzyme of roseoflavin biosynthesis the N, N-dimethyltransferase RosA, which generates roseoflavin from 8-demethyl-8-amino-riboflavin (AF). Consequently, the roseoflavin biosynthetic pathway depends on a phosphatase, which dephosphorylates AFP to AF. Here, we report on the identification and characterization of such an AFP phosphatase which we named RosC. The gene rosC is located immediately downstream of rosA and both genes are part of a cluster comprising 10 genes. Deletion of rosC from the chromosome of S. davaonensis led to reduced roseoflavin levels in the corresponding recombinant strain. In contrast to wild-type S. davaonensis, cell-free extracts of the rosC deletion strain did not catalyze dephosphorylation of AFP. RosC was purified from an overproducing Escherichia coli strain. RosC is the fastest enzyme of roseoflavin biosynthesis (k_cat 31.3 ± 1.4 min^–1). The apparent K_M for the substrate AFP was 34.5 µM. Roseoflavin biosynthesis is now completely understood––it takes three enzymes (RosB, RosC, and RosA) to convert the flavin cofactor riboflavin-5ʹ-phosphate into a potent antibiotic.     

Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene.

A gene encoding an alpha-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced. The alpha-1,2-mannosyltransferase which utilizes alpha-methylmannoside as acceptor of mannose from GDP-mannose was purified. The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides. Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA. A specific reaction product was used to screen a genomic library of S.cerevisiae. A fragment of approximately 5.7 kb was isolated, of which a 2.9 kb fragment was sequenced. It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified alpha-1,2-mannosyltransferase. The gene, designated MNT1, is located on the right arm of chromosome 4. It encodes a 442 amino acid polypeptide with a calculated mol. wt of 51.4 kDa. The corresponding mRNA has a length of approximately 1.6 kb. Overexpression of the MNT1 gene increased this alpha-1,2-mannosyltransferase activity approximately 2.5-fold. The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site. The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases. This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.     

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine–aldimine complex to a substrate–aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.     

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes.     

Purification,characterization, and cell wall localization of an α-fucosidase that inactivates a xyloglucan oligosaccharin

An α-fucosidase that releases fucosyl residues from oligosaccharide fragments of xyloglucan, a plant cell wall hemicellulosic polysaccharide, was purified to homogeneity from pea (Pisum sativum) epicotyls using a combination of cation exchange chromatography and isoelectric focusing. The α-fucosidase has a molecular mass of 20 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The α-fucosidase has an isoelectric point of 5.5. The substrate specificity of the α-fucosidase was determined by high performance anion exchange chromatographic analysis of oligosaccharide substrates and products. The enzyme hydrolyzes the terminal α-1,2-fucosidic linkage of oligosaccharides and does not cleave p-nitrophenyl-α-L-fucoside. The enzyme does not release measurable amounts of fucosyl residues from large polysaccharides. The subcellular localization of α-fucosidase in pea stems and leaves has been studied by immunogold cytochemistry. The α-fucosidase accumulates in primary cell walls and is not detectable in the middle lamella or in the cytoplasm of 8-day-old stem tissue and 14-day-old leaf tissue. α-Fucosidase activity was readily detected in extracts of 8-day-old stem tissue. No significant α-fucosidase acitivity or immunogold labeling of the α-fucosidase was detected in 2- and 4-day-old stem tissue indicating that production of α-fucosidase is developmentally regulated.     

Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis

Levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis, was cloned and functionally characterized. A Ginkgo biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl diphosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme.     

1,2‐Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway

Treponema denticola synthesizes phosphatidylcholine through a licCA‐dependent CDP‐choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP‐choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2‐diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP‐choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA‐Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2‐diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host‐associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.     

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures

The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k(3) values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H(2)O(2). The results also showed that sites for monolignol beta-O-4 cross-coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.     

Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters

The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esterified to an alcohol of a similar length. As part of an EST project, we recently identified an acyltransferase with 19% sequence identity (amino acid) to a bacterial ‘bifunctional’ wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deficient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in VLCFA esters of methyl, isoamyl, and short-to-medium straight chain alcohols (C4–C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and ¹⁴C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be sufficient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the first characterization of a eukaryotic protein from the WS/DGAT family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning, characterization, and downregulation of an acyltransferase that catalyzes the malonylation of flavonoid and naphthol glucosides in tobacco cells

Tobacco cells (Nicotiana tabacum L. Bright Yellow T-13) exposed to harmful naphthols accumulate them as glucosylated and further modified compounds [Taguchi et al. (2003a) Plant Sci. 164, 231-240]. In this study, we identified the accumulated compounds to be 6'-O-malonylated glucosides of naphthols. Cells treated with various phenolic compounds accumulated the flavonoids mainly as malonylglucosides. To clarify the function of this malonylation in tobacco, we isolated the cDNA encoding a malonyltransferase (NtMaT1) from a cDNA library derived from tobacco cells. The heterologous expression of the gene in Escherichia coli revealed that the recombinant enzyme had malonyltransferase activity against several phenolic glucosides such as flavonoid 7-O-glucosides, flavonoid 3-O-glucosides and naphthol glucosides. The substrate preference of the enzyme was similar to that of the tobacco cell extract. Malonylation activity in the transgenic cells markedly decreased with the suppression of the expression of NtMaT1 mRNA in tobacco BY-2 cells by RNA interference. The compounds administered to the transgenic cells were accumulated in the cells as glucosides or other modified compounds in place of malonylglucosides. These results show that NtMaT1 is the main catalyst of malonylation on glucosides of xenobiotic flavonoids and naphthols in tobacco plants.     

Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved in aflatoxin biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5'-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5'-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.     

The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor. Isolation, cloning, heterologous expression, and substrate specificity

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form by O-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicolor employing Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea mays flavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenous p-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared with p-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.     

Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.     

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.     

Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol

The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes.     

Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides

A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.