The N-glycan acceptor specificity of a glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope

The acceptor specificity of a rat brain glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope on glycoproteins, was investigated using asialoorosomucoid as a model acceptor substrate. Structural analysis of N-linked oligosaccharides, to which glucuronic acid was transferred by GlcAT-P, by means of two-dimensional mapping of pyridylamino-oligosaccharides and MS spectrometry, demonstrated that the enzyme transferred glucuronic acid to bi-, tri-, and tetra-antennary complex type sugar chains, with almost equal efficiency, indicating that the enzyme has no preference as to the number of acceptor sugar branches. Next, we studied the branch specificity of this enzyme by means of the selective branch scission method involving two step exoglycosidase digestion using authentic pyridylamino-oligosaccharides. The GlcAT-P is highly specific for the terminal N-acetyllactosamine structure and no glucuronic acid was incorporated into a Gal1-3GlcNAc moiety. The GlcAT-P transferred glucuronic acid to the galactose residues in the N-acetyllactosamine branches of bi-, tri-, and tetra-antennary oligosaccharide chains, with different efficiencies and most preferentially to those in the Gal1-4GlcNAc1-4Man1-3 branch.     

Different acceptor specificities of two glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate

The biosynthesis of HNK-1 carbohydrate is mainly regulated by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase (HNK-1 ST). To determine how the two glucuronyltransferases are involved in the biosynthesis of the HNK-1 carbohydrate, we prepared soluble forms of GlcAT-P and GlcAT-S fused with the IgG-binding domain of protein A and then compared the enzymatic properties of the two enzymes. Both GlcAT-P and GlcAT-S transferred glucuronic acid (GlcA) not only to a glycoprotein acceptor, asialoorosomucoid (ASOR), but also to a glycolipid acceptor, paragloboside. The activity of GlcAT-P toward ASOR was enhanced fivefold in the presence of sphingomyelin, but there were no effects on that of GlcAT-S. The activities of the two enzymes toward paragloboside were only detected in the presence of phospholipids such as phosphatidylinositol. Kinetic analysis revealed that the K(m) value of GlcAT-P for ASOR was 10 times lower than that for paragloboside. Furthermore, acceptor specificity analysis involving various oligosaccarides revealed that GlcAT-P specifically recognized N-acetyllactosamine (Galbeta1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognized not only the terminal Galbeta1-4GlcNAc structure but also the Galbeta1-3GlcNAc structure and showed the highest activity toward triantennary N-linked oligosaccharides. GlcAT-P transferred GlcA to NCAM about twice as much as to ASOR, whereas GlcAT-S did not show any activity toward NCAM. These lines of evidence indicate that these two enzymes have significantly different acceptor specificities, suggesting that they may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system.     

Purification and characterization of two recombinant human glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate in Escherichia coli

Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate, which is spatially and temporally regulated in the nervous system. To clarify the enzymatic properties of the respective glucuronyltransferases, we established an expression system for producing large amounts of soluble forms of flag-tagged human GlcAT-P and GlcAT-S in Escherichia coli. Approximately 15 and 6 mg of enzymatically active flag-GlcAT-P and flag-GlcAT-S were purified from E. coli cells in 5 liters of culture medium, respectively. These recombinant enzymes transferred GlcA to a glycoprotein acceptor, asialo-orosomucoid (ASOR), as well as a glycolipid acceptor, paragloboside. The specific activity of the recombinant GlcAT-P (1100 nmol/min/mg) toward a glycoprotein acceptor, ASOR, was comparable to that of the enzyme (4300 nmol/min/mg) purified from rat brain. Phosphatidylinositol (PI) is specifically required for expression of the activity of the recombinant enzymes toward a glycolipid acceptor, paragloboside. The recombinant GlcAT-P was highly specific for the terminal type II structure, Galbeta1-4GlcNAc, while the recombinant GlcAT-S recognized not only the type II structure, Galbeta1-4GlcNAc, but also the type I structure, Galbeta1-3GlcNAc. These acceptor specificities were similar to those of the native enzymes.     

Mice deficient in nervous system-specific carbohydrate epitope HNK-1 exhibit impaired synaptic plasticity and spatial learning

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.     

Comparative rates of transfer of N-acetylneuraminic acid to acceptors bearing one or more Gal(beta 1-4)GlcNAc terminus by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase from embryonic chicken liver. Utilization of oligosaccharides as acceptors in sialyltransferase assays.

Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.     

The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.     

Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.     

Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in processing a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8-A resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designated UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicates human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.     

A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor

This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioure-idoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low Km values and Vmax values comparable in magnitude (15-100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver alpha 2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.     

Molecular cloning and functional characterization of a Lepidopteran insect beta4-N-acetylgalactosaminyltransferase with broad substrate specificity, a functional role in glycoprotein biosynthesis, and a potential functional role in glycolipid biosynthesis

A degenerate PCR approach was used to isolate a lepidopteran insect cDNA encoding a beta4-galactosyl-transferase family member. The isolation and initial identification of this cDNA was based on bioinformatics, but its identification as a beta4-galactosyltransferase family member was experimentally confirmed. The newly identified beta4-galactosyltransferase family member had unusually broad donor and acceptor substrate specificities in vitro, as transferred galactose, N-acetylglucosamine, and N-acetylgalactosamine to carbohydrate, glycoprotein, and glycolipid acceptors. However, the enzyme preferentially utilized N-acetylgalactosamine as the donor for all three acceptors, and its derived amino acid sequence was closely related to a known N-acetylgalactosaminyltransferase. These data suggested that the newly isolated cDNA encodes a beta4-N-acetylgalactosaminyltransferase that functions in insect cell glycoprotein biosynthesis, glycolipid biosynthesis, or both. The remainder of this study focused on the role of this enzyme in N-glycoprotein biosynthesis. The results showed that the purified enzyme transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to a synthetic N-glycan in vitro. The structure of the reaction product was confirmed by chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses. Co-expression of the new cDNA product in insect cells with an N-glycoprotein reporter showed that it transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to this N-glycoprotein in vivo. Confocal microscopy showed that a GFP-tagged version of the enzyme was localized in the insect cell Golgi apparatus. In summary, this study demonstrated that lepidopteran insect cells encode and express a beta4-N-acetylgalactosaminyltransferase that functions in N-glycoprotein biosynthesis and perhaps in glycolipid biosynthesis, as well. The isolation and characterization of this gene and its product contribute to our basic understanding of insect protein N-glycosylation pathways and to the growing body of evidence that insects can produce glycoproteins with complex N-glycans.     

Glucose-1-phosphotransferase and N-acetylglucosamine-1-phosphotransferase have distinct acceptor specificities

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to endoglycosidase H-sensitive oligosaccharides on acceptor glycoproteins. We have previously demonstrated that Glc-phosphotransferase was specific for UDP-Glc as its nucleotide sugar substrate and thus appeared to be distinct from UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase), an enzyme specific for lysosomally destined acceptor glycoproteins. Here, sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs of endogenous acceptor glycoproteins in embryonic chick neural retina homogenates labeled by the presence of [beta-32P]UDP-Glc were shown to be distinct from those labeled by [beta-32P]UDP-GlcNAc, indicating that the two enzymatic activities recognize different populations of endogenous glycoproteins. To further probe the acceptor specificities of these enzymes, three glycoproteins known to be exogenous acceptors for GlcNAc-phosphotransferase were included in assays for Glc-phosphotransferase from retinal homogenates. Cathepsin D and beta-N-acetylhexosaminidase had no significant effects on phosphoglucose incorporation. Uteroferrin, an acid phosphatase, had a pronounced inhibitory effect on incorporation from UDP-Glc, and subsequent experiments suggested that phosphorylation of the Glc-phosphotransferase or another protein may be necessary for maximal activity to be seen. Also, I-cells, which have previously been shown to possess no GlcNAc-phosphotransferase activity, and control human fibroblasts were assayed for both Glc-phosphotransferase and GlcNAc-phosphotransferase. GlcNAc-phosphotransferase activity was observed only in control cells, whereas Glc-phosphotransferase was observed in both I-cells and controls at similar specific activities.     

Cloning and chromosomal mapping of human glucuronyltransferase involved in biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate is expressed on various cell adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substrate interactions. Here we describe the isolation of a cDNA encoding human glucuronyltransferase (GlcAT-P), which is a key enzyme in the biosynthesis of the HNK-1 carbohydrate. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein of 334 amino acids. Human GlcAT-P was 98.2% identical with rat GlcAT-P in amino acid sequence, the exception being the length of the cytoplasmic tail. Northern blot analysis indicated that human GlcAT-P is expressed mainly in the brain. There is a single copy of the human GlcAT-P gene (HGMW-approved symbol B3GAT1), and it was mapped to chromosome 11q25.     

Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose.

Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.     

UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds

We detected UDP-glucuronic acid:soyasapogenol glucuronosyltransferase (UGASGT) activity in the microsomal fraction from germinating soybean (Glycine max [L.] Merr.) seed. A microsomal fraction was isolated from germinating soybean seed and treated with various detergents to solubilize the enzyme. UGASGT activity was monitored throughout purification using UDP-[U-(14)C]glucuronic acid and soyasapogenol B as substrates. Purification of UGASGT was achieved by HiTrap Q, Superdex 200, and HiTrap Blue chromatography procedures. This resulted in >205-fold enrichment relative to the starting homogenate. UGASGT was found to require divalent cations for activity. Studies on the substrate specificity of UGASGT demonstrated that the specificity for the sugar residue transferred was very high, as activity was scarcely found when UDP-glucuronic acid was replaced by other UDP sugars: UDP-glucose and UDP-galactose. Soyasapogenols, which are the aglycons of soybean saponin, are usable acceptors, but glycyrrhetinic acid, sophoradiol, beta-amyrin, and flavonoids are not. These findings suggest that this UGASGT was a specific enzyme for UDP-glucuronic acid as a donor and soyasapogenols as acceptors, and that it was related to the biosynthesis of the sugar chain in soybean saponin. This study provides a basis for the molecular characterization of a key enzyme in saponin biosynthesis in soybean. The isolation of the gene may enable its use in the elucidation of the biosynthesis and physiological role of saponins in soybean.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope

A cDNA encoding a novel glucuronyltransferase was cloned from a rat brain cDNA library. The cDNA sequence contained an open reading frame encoding 324 amino acids, with type II transmembrane topology. The amino acid sequence revealed 49% homology to rat GlcAT-P, a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins, [Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098] and the highest sequence homology was found in the catalytic region. Northern blot analysis indicated that this newly cloned glucuronyltransferase is expressed in the nervous system, consistent with the selective localization of the HNK-1 carbohydrate epitope in the nervous system. Transfection of this cDNA into COS-1 cells induced the expression of the HNK-1 carbohydrate epitope on cell surfaces, and induced the morphological changes in these cells. These results indicated that this newly cloned cDNA is a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.     

Enzymes of the glucuronic acid pathway in Wistar and Gunn rats and their heterozygotes

A significant difference in UDP glucuronyltransferase activity (with p-nitrophenol as an acceptor) was found in the liver and kidneys of homozygous Wistar and Gunn rats. There was also a significant difference in hepatic UDP glucuronyltransferase activity between homozygous Wistar and heterozygous Gunn rats when the enzyme preparations were first activated by adding surfactants to the reaction mixture. This determination of surfactant-activated UDP glucuronyltransferase can be used to distinguish Wistar rats from heterozygous Gunn rats. Other enzymes of the glucuronic acid pathway were also studied in the liver and kidneys of homozygous Wistar and Gunn rats, but no differences were found.This study has been supported by grants from the U.S. Public Health Service (AM-06018-09 and the National Research Council for Natural Sciences, Finland.