Murine Enzyme Research Articles

Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity

Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, has been identified to show anti-inflammatory, anti-viral and anti-cancer activities. The present study, therefore, examined effects of CAPE on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with CAPE significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ). CAPE also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-κB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene, revealed that CAPE inhibited the iNOS promoter activity induced by LPS plus IFN-γ through the NF-κB sites of the iNOS promoter. In addition, CAPE directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level through the suppression of NF-κB activation, and by directly inhibiting the catalytic activity of iNOS.

Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

Expression and Characterization of a Murine Enzyme Able to Cleave β-Carotene: THE FORMATION OF RETINOIDS

Because animals are not able to synthesize retinoids de novo, ultimately they must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (beta-carotene 15,15'-dioxygenase) has been characterized primarily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encoding a mouse homolog of this enzyme. When the cDNA was expressed in either Escherichia coli or Chinese hamster ovary cells, expression conferred upon bacterial and Chinese hamster ovary cell homogenates the ability to cleave beta-carotene to retinal. Several lines of evidence obtained upon kinetic analyses of the recombinant enzyme suggested that carotene cleavage enzyme interacts with other proteins present within cell or tissue homogenates. This was confirmed by pull-down experiments upon incubation of recombinant enzyme with tissue 12,000 x g supernatants. Matrix-assisted laser desorption ionization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate dehydrogenase associates with recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain reaction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understanding the biochemistry of carotene cleavage to retinoids.

Identification, tissue expression, and chromosomal position of a novel gene encoding human ubiquitin-conjugating enzyme E2-230k

Through large-scale human genome mapping and sequencing of a region at chromosome 17q25.1 that is of particular interest because genes associated with breast, ovarian, and esophageal cancer are likely to be located there, we isolated the cDNA of a novel member of the family of ubiquitin-conjugating enzymes. The predicated peptide showed 97% identity in amino-acid sequence to murine ubiquitin-conjugating enzyme E2-230k (Mus UBE2-230k). The human cDNA consisted of 4878 nucleotides with an open reading frame encoding 1138 amino acids; the ∼5 kb transcript was expressed predominantly in skeletal muscle and heart. The predicted UBE2-230k peptide contained a motif typical of the UBC domain that has been implicated in the ubiquitin-dependent proteolytic system and related pathways.

Acetyl-CoA Synthetase 2, a Mitochondrial Matrix Enzyme Involved in the Oxidation of Acetate

Using peptide sequences derived from bovine cardiac acetyl-CoA synthetase (AceCS), we isolated and characterized cDNAs for a bovine and murine cardiac enzyme designated AceCS2. We also isolated a murine cDNA encoding a hepatic type enzyme, designated AceCS1, identical to one reported recently (Luong, A., Hannah, V. C., Brown, M. S., and Goldstein, J. L. (2000) J. Biol. Chem. 275, 26458-26466). Murine AceCS1 and AceCS2 were purified to homogeneity and characterized. Among C2-C5 short and medium chain fatty acids, both enzymes preferentially utilize acetate with similar affinity. The AceCS2 transcripts are expressed in a wide range of tissues, with the highest levels in heart, and are apparently absent from the liver. The levels of AceCS2 mRNA in skeletal muscle were increased markedly under ketogenic conditions. Subcellular fractionation revealed that AceCS2 is a mitochondrial matrix enzyme. [(14)C]Acetate incorporation indicated that acetyl-CoAs produced by AceCS2 are utilized mainly for oxidation.

Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution

Sphingosine-1-phosphate (SPP), the product of sphingosine kinase, is an important signaling molecule with intra- and extracellular functions. The cDNA for the mouse sphingosine kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human sphingosine kinase (huSPHK1). Sequence analysis comparison revealed that this kinase is evolutionarily very conserved, having a high degree of homology with the murine enzyme, and presenting several conserved regions with bacteria, yeast, plant, and mammalian proteins. Expressed huSPHK1 cDNA specifically phosphorylates d- erythro-sphingosine and, to a lesser extent, d,l- erythro-dihydrosphingosine, and not at all the ‘ threo’ isoforms of dihydrosphingosine; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-phosphate (PIP); or phosphatidylinositol-4,5-bisphosphate (PIP 2). huSPHK1 shows typical Michaelis–Menten kinetics ( V max=56 μM and K m=5 μM). The kinase is inhibited by d,l- threo-dihydrosphingosine ( K i=3 μM), and by N, N- dimethyl-sphingosine ( K i=5 μM). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respectively. It is also expressed in brain and heart. In addition, database searches with the stSG2854 sequence indicate that huSPHK1 is also expressed in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

Recognition of a Sequestered Self Peptide by Influenza Virus-Specific CD8+ Cytolytic T Lymphocytes

The Ag receptors on CD8+ CTL recognize foreign antigenic peptides associated with cell surface MHC class I molecules. Peptides derived from self proteins are also normally presented by MHC class I molecules. Here we report that an H-2Kd-restricted murine CD8+ CTL clone directed to an influenza hemagglutinin epitope can recognize a peptide derived from the murine mitochondrial aconitase enzyme in association with H-2Kd molecules. Surprisingly, this self peptide is not normally displayed on the cell surface associated with the restricting MHC class I molecule. Several lines of evidence suggest that this self peptide, although requiring association with the Kd molecule for CTL recognition, is not associated with this or other MHC class I allele under physiologic conditions in intact cells. Rather, it is sequestered in the cytoplasm associated with a carrier protein and is released only upon cell disruption. These results suggest a means of restricting the entry of self peptide into the class I pathway. In addition, this finding raises the possibility that self peptides sequestered within the cell can, after release from damaged cells, interact with MHC class I molecules on bystander cells and trigger autoimmune injury by virus-specific CTLs during viral infection.

Mammalian 5′(3′)-Deoxyribonucleotidase, cDNA Cloning, and Overexpression of the Enzyme in Escherichia coli and Mammalian Cells

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

Effects of Long-Term Vitamin E and Butylated Hydroxytoluene Supplemented Diets on Murine Intestinal and Hepatic Antioxidant Enzyme Activities

Effects of Long-Term Vitamin E and Butylated Hydroxytoluene Supplemented Diets on Murine Intestinal and Hepatic Antioxidant Enzyme Activities

Bile acid structure and selective modulation of murine hepatic cytochrome P450-linked enzymes.

We examined the effects of the administration of different bile acids on in vivo hepatic murine cytochrome P450 (CYP) content, nicotinamide adenine dinucleotide phosphate (NADPH)-CYP-reductase, and individual mixed-function oxidases (MFOs). Neither CYP level nor reductase were appreciably affected by single intraperitoneal administration of taurodeoxycholic acid (TDCA) (12.2 or 24.4 mg x kg(-1) bw). MFO to various isoenzymes were slightly reduced 24 hours after treatment. Taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) both induced CYP, reductase, and MFOs. CYP3A1/2-linked activity (i.e., testosterone 6beta-hydroxylase, and N-demethylation of aminopyrine) in a dose-dependent fashion was enhanced ( approximately 2-3-fold). CYP2E1- (hydroxylation of p-nitrophenol), CYP1A2-(O-demethylation of methoxyresorufin), CYP2A1/2- and CYP2B1/2-(6alpha-hydroxylase), and CYP2B9- (16alpha-hydroxylase) dependent MFOs, as well as 7alpha-, 16beta-, 2alpha-, and 2beta-hydroxylations, were all significantly induced by THDCA. Apart from alkoxyresorufin metabolism and a modest CYP2E1 increase, TUDCA behaved like THDCA. A generalized induction was also recorded after ursodeoxycholic acid (UDCA) administration. THDCA and TDCA did not show substantial differences in the N-demethylation of aminopyrine when different species (rat vs. mouse) and administration route (intraperitoneal vs. intravenous) were compared. Results on the most affected isoenzymes, CYP3A1/2 (THDCA, TUDCA, and UDCA) and CYP2E1 (UDCA), were sustained by means of Western immunoblotting. CYP3A induction was paralleled by a corresponding increase in mRNA. These data could partially explain the therapeutic mechanism of UDCA, TUDCA, and THDCA in chronic cholestatic liver disease. CYP3A induction, which is linked to P-glycoprotein (Pgp) family overexpression, may enhance hepatic metabolism, transport, and excretion of toxic endogenous lipophilic bile acids.

Molecular Cloning and Tissue Expression of the Murine Analog to Human Stratum Corneum Chymotryptic Enzyme

Human stratum corneum chymotryptic enzyme (SCCE) may play a central part in epidermal homeostasis. Its proposed function is to catalyze the degradation of intercellular structures, including desmosomes, in the stratum corneum as part of the desquamation process. In order to facilitate physiologic and pathophysiologic studies on SCCE we have looked for the corresponding murine enzyme. A cDNA obtained by reverse transcription-polymerase chain reaction with total RNA prepared from mouse tails as starting material was cloned, and the expression of the corresponding mRNA studied. The murine cDNA showed 77% homology to human SCCE cDNA. It had an open-reading frame encoding a protein comprising 249 amino acids with 82% amino acid sequence homology to human SCCE including the conserved sequences of the catalytic traid of mammalian serine proteases. The murine protein was deduced to have a 21 amino acid signal peptide and a four amino acid propeptide ending with a tryptic cleavage site, followed by a sequence motif identical to the N-terminal amino acid sequence of native active human SCCE. As in human SCCE the P2 position of the propeptide was occupied by an acidic amino acid residue, and the position corresponding to the suggested bottom of the primary substrate specificity pouch occupied by an asparagine residue. Analyses of mouse tissues by reverse transcriptase-polymerase chain reaction showed high expression in the skin, low expression in lung, kidney, brain, heart, and spleen, and no expression in liver or skeletal muscle. In situ hybridization of mouse skin showed expression in high suprabasal keratinocytes and in the luminal parts of hair follicles. Our results strongly suggest that we have cloned the murine analog of human SCCE cDNA.

Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays.

Two strategies are described for detecting constitutive or induced protein-protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP (FK506 binding protein) and FRAP (FKBP-rapamycin binding protein) fused to DHFR fragments and show a rapamycin dose-dependent survival of clones that requires approximately 25 molecules of reconstituted DHFR per cell. A fluorescence assay also is described, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Formation of the FKBP-rapamycin-FRAP complex is detected in stably and transiently transfected cells. Quantitative rapamycin dose-dependence of this complex is shown to be consistent with in vitro binding and distinguishable from a known constitutive interaction of FKBP and FRAP. We also show that this strategy can be applied to study membrane protein receptors, demonstrating dose-dependent activation of the erythropoietin receptor by ligands. The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both. High-throughput drug screening and quantitative analysis of induction or disruption of protein-protein interactions are also made possible.

Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide.

Leflunomide is currently in phase-III clinical trials for the treatment of rheumatoid arthritis. In this study, we have focused our efforts on the study of the mechanism of action of the active metabolite of leflunomide, A77 1726, in cells and tissue of human origin. The human high-affinity binding protein for radiolabelled A77 1726 was purified from solubilized U937 membranes by following the binding activity through the purification process and was characterized as the mitochondrial enzyme dihydro-orotate dehydrogenase (DHO-DH). The human and murine enzyme displayed identical pI and molecular mass values on SDS/PAGE (43 kDa), which contrasts notably with previous reports suggesting a molecular mass of 50 kDa for the human enzyme. DHO-DH activity was inhibited by A77 1726 and its analogue HR325 with similar potency in U937 and human spleen membrane preparations. HR325 was found to be anti-proliferative for phytohaemagglutinin-stimulated human peripheral blood mononuclear cells, at the same concentrations that caused accumulation of DHO and depletion of uridine. Supplementation of the cultures with exogenous uridine led to partial abrogation of the anti-proliferative effect. This is in line with our recent demonstration that the anti-proliferative effect in vitro of A77 1726 on lipopolysaccharide-stimulated mouse spleen cells was mediated by DHO-DH inhibition [Williamson, Yea, Robson, Curnock, Gadher, Hambleton, Woodward, Bruneau, Hambleton, Moss et al., (1995) J. Biol. Chem. 270, 22467-22472]. Thus, DHO-DH inhibition by A77 1726 and its analogues is responsible for the anti-proliferative effects in vitro of the compounds on human cells and is likely to be responsible for some of its effects in vivo.

Caenorhabditis elegansContains Two Distinct Acid Sphingomyelinases

Mounting evidence supports a role for acid sphingomyelinase (ASM) in cellular stress signaling. Only murine and human sphingomyelinases have been defined at the molecular level. These enzymes are the products of a conserved gene and at the amino acid level share 82% identity. In this study, we show that the nematode Caenorhabditis elegans possesses two ASMs, termed ASM-1 and ASM-2 encoded by two distinct genes, but lacks detectable neutral sphingomyelinase activity. The C. elegans ASMs are about 30% identical with each other and with the human and murine enzymes. The conserved regions include a saposin-like domain, proline-rich domain, and a putative signal peptide. In addition, 16 cysteines distributed throughout the molecules, and selected glycosylation sites, are conserved. The expression of these genes in C. elegans is regulated during development. Asm-1 is preferentially expressed in the embryo, whereas asm-2 is predominantly expressed in postembryonic stages. When transfected as Flag-tagged proteins into COS-7 cells, ASM-1 is found almost entirely in a secreted form whereas only 20% of ASM-2 is secreted. Only the secreted forms display enzymatic activity. Furthermore, ASM-2 requires addition of Zn2+ to be fully active, whereas ASM-1 is active in the absence of cation. C. elegans is the first organism to display two ASMs. This finding suggests the existence of an ASM gene family.

DNA binding discrimination of the murine DNA cytosine-C5 methyltransferase

Mammalian DNA cytosine-C5 methyltransferase modifies the CpG dinucleotide in the context of many different genomic sequences. A rigorous DNA binding assay was developed for the murine enzyme and used to define how sequences flanking the CpG dinucleotide affect the stability of the enzyme:DNA complex. Oligonucleotides containing a single CpG site form reversible 1:1 complexes with the enzyme that are sequence-specific. A guanine/cytosine-rich 30 base-pair sequence, a mimic of the GC-box cis-element, bound threefold more tightly than an adenine/thymine-rich sequence, a mimic of the cyclic AMP responsive element. However, the binding discrimination between hemi- and unmethylated forms of these DNA substrates was small, as we previously observed at the KmDNA level (Biochemistry, 35, 7308–7315 (1996)). Single-stranded substrates are bound much more weakly than double-stranded DNA forms.An in vitro screening method was used to select for CpG flanking sequence preferences of the DNA methyltransferase from a large, divergent population of DNA substrates. After five iterative rounds of increasing selective pressure, guanosine/cytosine-rich sequences were abundant and contributed to binding stabilization for at least 12 base-pairs on either side of a central CpG. Our results suggest a read-out of sequence-dependent conformational features, such as helical flexibility, minor groove dimensions and critical phosphate orientation and mobility, rather than interactions with specific bases over the course of two complete helical turns. Thus, both studies reveal a preference for guanosine/cytosine deoxynucleotides flanking the cognate CpG. The enzyme specificity for similar sequences in the genome may contribute to the in vivo functions of this vital enzyme.

Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin.

The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2 alpha, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2 alpha demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2 alpha is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2 alpha to these inhibitors suggests that their use should be reevaluated in the study of PI3-Ks.

Selective prostaglandin G/H synthase (PGHS)-2 inhibitors show greater inhibitory activities on human PGHS-2 than on murine PGHS-2 in intact cells

Excess eicosanoid formation during inflammation has been attributed to the expression of the gene coding for the inducible isoform of prostaglandin G/H synthase (PGHS-2). Human and murine PGHS-2 proteins differ in 73 out of the 604 amino acids. When comparing the inhibitory effects of a panel of PGHS-inhibitors in a whole cell human and murine PGHS-2 assay carried out under identical conditions, classical NSAIDs with the exception of aspirin and tenoxicam showed similar inhibitory effects on both human and murine PGHS-2 enzymes. However, the PGHS-2 selective inhibitors nimesulide, flosulide and NS398 showed a much greater inhibition of human PGHS-2. We suggest that these differences could be due to the genetic differences of human and murine PGHS-2.

Cloning, expression and characterisation of murine procathepsin E

The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli. The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors. The majority of the recombinant enzyme was present as a homodimer (M r ∼80) in which the two monomers were linked by an intermolecular disulfide bond. By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N-terminus of the mature proteinase. The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full-length cDNA now enables structural investigations and attempts to generate `knock-out' mice deficient in this important aspartic proteinase to be undertaken.

Stably transfected human cells overexpressing rat brain endopeptidase 3.4.24.16: biochemical characterization of the activity and expression of soluble and membrane-associated counterparts.

We recently cloned endopeptidase-24.16 (neurolysin; EC 3.4.24.16), a neurotensin-degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3-lambda 7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase-24.16 immunoreactivity and exhibited QFS- and neurotensin-hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane-associated form of endopeptidase-24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase-24.16. The membrane-associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the "neuronal" phenotype exhibited by the enzyme in transfected cells.

Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera.

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.