©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction, Localization, and Purification of a Novel Sialidase, Deaminoneuraminidase (KDNase), from Sphingobacterium multivorum(*)

(Received for publication, September 21, 1995)

Satoru Nishino (1) Hidehito Kuroyanagi (1) Takaho Terada (1) Sadako Inoue (2) Yasuo Inoue (1) Frederic A. Troy (3) Ken Kitajima (1)(§)

From the  (1)Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan, the (2)School of Pharmaceutical Sciences, Showa University, Hatanodai-1, Tokyo 142, Japan, and the (3)Department of Biological Chemistry, University of California School of Medicine, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDNalpha23Galbeta13GalNAcalpha13[KDNalpha2 (8KDNalpha2)6]GalNAcol, as a sole carbon source induced KDNase SM activity 15-40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to determine the molecular mechanism whereby the enzyme can discriminate between KDN and N-acylneuraminic acid.


INTRODUCTION

The natural occurrence of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), (^1)deaminated neuraminic acid, was first discovered in 1986 by Nadano et al.(1) . Subsequently, an increasing number of other KDN-glycoconjugates have been reported(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . Substitution of the aminoacyl group at C-5 on Neu5Ac or Neu5Gc by a hydroxyl group blocks almost completely the action of bacterial exosialidases that are commonly used for the identification and structure-function studies of sialoglycoconjugates (1, 8, 15) .

Recently, we isolated and identified a Gram-negative soil bacterium, Sphingobacterium multivorum, that produced a new sialidase that selectively catalyzed the hydrolysis of different types of KDN-ketosidic linkages(16) . The partial purification of this enzyme, designated KDNase SM, and its substrate specificity were also described previously(16) . The most important feature of KDNase SM was the complete absence of sialidase activity that could release Neu5Ac and Neu5Gc residues from a variety of naturally occurring Neu5Ac- and Neu5Gc-containing glycoconjugates. This unique specificity was in distinct contrast with other eukaryotic KDN-sialidases that hydrolyzed both KDN and N-acylneuraminic acid alpha-ketosidic linkages (17, 18) . Since the first report of KDN residues in fish egg polysialoglycoproteins(1) , it has been presumed that KDN residues would not likely be restricted in occurrence to only lower vertebrates but rather would also be expressed on mammalian cells. With the recent findings that KDN-glycoconjugates are implicated in fertilization in fish egg polysialoglycoproteins(4, 7, 8) , it became important to determine the extent of the natural occurrence of KDN-glycoconjugates in other eukaryotic organisms. While anti-KDN antibodies have been used to show that KDN-glycoconjugates are expressed in rat pancreas(12) , confirmation of this conclusion has awaited the availability of a KDNase specific for the hydrolysis of KDN-ketosidic linkages but not N-acylneuraminyl linkages. Indeed, we have shown recently that KDNase SM can be used in combination with the mAbbulletkdn8kdn-reactive antibodies as a diagnostic enzyme for the specific detection of KDN-glycotopes in a variety of different mammalian cells. (^2)

The above observations raise the interesting question as to how KDNase SM can discriminate between KDN and N-acylneuraminic acid residues. Attempts to answer this question have been frustrated by the limited availability of KDNase SM. Because of the importance and interest in using KDNase in future investigations, we initiated studies to isolate and purify large quantities of KDNase SM. This report describes that KDNase SM is an inducible enzyme in S. multivorum when the cells are grown in the presence of KDN-oligosaccharide alditols (KDN-OS), KDNalpha2-3Galbeta1-3GalNAcalpha1-3[KDNalpha2-(8KDNalpha2-)-6]GalNAcol as the sole carbon source. We also show that the enzyme is localized in the periplasm. Based on these observations, we were successful in purifying the inducible enzyme 700-fold after osmotic shock, to homogeneity.


EXPERIMENTAL PROCEDURES

Bacteria

Isolation and growth of a strain of Sphingobacterium multivorum that could grow on KDN-oligosaccharide alditols as the sole carbon source were carried out as described previously(16) .

Assay for KDNase Activity

4-Methylumbelliferyl KDN (4-MU-KDN) was used as a substrate to quantitate KDNase activity. 4-MU-KDN was kindly provided by Dr. T. G. Warner (Genentech, Inc.) (17) . Enzyme assay mixtures containing 1.6 µl of 4-MU-KDN (0.71 µg; 1.6 nmol) were incubated in a total volume of 21.6 µl with 20 µl of enzyme dissolved in 100 mM Tris acetate buffer, pH 6.0, containing 100 mM NaCl at 25 °C. After a 30-min incubation, 3.0 ml of 85 mM glycine carbonate buffer, pH 9.3, were added to a 20-µl aliquot of each incubation mixture. The 4-methylumbelliferone released was determined fluorimetrically ( = 365 nm and = 450 nm) with a JASCO 821-FP fluorescence spectrophotometer as described by Warner and O'Brien(19) . Control incubations were carried out without enzyme. One unit of enzyme activity is defined as the amount of enzyme that catalyzed the hydrolysis of 1 nmol of 4-MU-KDN per min at 25 °C.

Assay for Endoglycosidase, Exoglycosidase, and Protease Activities

The purified KDNase was assayed for the presence of contaminating protease, exo- and endoglycosidase, and peptide:N-glycanase activities as described previously (16) .

Preparation of Inducer Oligosaccharides

KDN and KDN-OS, KDNalpha2-3Galbeta1-3GalNAcalpha1-3[KDNalpha2-(8KDNalpha2-)(n)-6]GalNAcol with n = 5, were prepared as described previously(16) . A KDN-oligosaccharide alditol-rich fraction, designated ``enriched'' KDN-OS, was prepared as follows: rainbow trout ovarian fluid (12.3 liters) was concentrated and lyophilized, and 110 g of dried powder were obtained. The lyophilized powder (50 g) was extracted once with 1.0 liter of chloroform/methanol (2:1 (v/v)) and then with 1.0 liter of chloroform/methanol (1:2 (v/v)) at room temperature for 2 h(11) . The delipidated residue was air-dried and weighed 39 g. The delipidated ovarian fluid-derived powder (10 g) was suspended in 100 ml of 1 M NaBH(4), 0.1 M NaOH and incubated at 37 °C with agitating. After 24 h, 50 ml of the same alkaline borohydride solution were added and incubated for another 24 h. The reaction mixture was centrifuged at 9,000 times g for 20 min, and the supernatant was desalted by passage through a Sephadex G-25 column (2.0 times 150 cm, eluted with water) after neutralization to pH 6 with glacial acetic acid followed by concentration to 50 ml. The desalted fraction was enriched in KDN-oligosaccharide alditols. The amount of KDN was quantitated by the TBA method(20) .

Induction of KDNase SM Activity in S. multivorum

40 ml of M9 mineral medium containing (in grams/liter) Na(2)HPO(4), 6.0 g; KH(2)PO(4), 3.0 g; NH(4)Cl, 1.0 g; NaCl, 0.5 g; MgSO(4), 1 mmol; CaCl(2), 0.1 mmol; and 1% (w/v) of casamino acids and glucose were inoculated with 4 times 10^8 cells of S. multivorum and incubated at 25 °C. Cells in late log or early stationary phase of growth (42-46 h) were harvested and washed twice with M9 medium. Washed cells (6.1 times 10) were inoculated into 2.0 ml of M9 medium with or without either 0.1% (w/v) enriched KDN-OS, KDN-OS, KDN, Neu5Ac, mild acid hydrolysate of colominic acid (oligoNeu5Ac)(21) , or Glc and incubated at 25 °C for times varying between 6 and 48 h. At each time point, viable cell numbers and KDNase activity were determined. Cell numbers were determined by counting colonies that grew up from a series of diluted cell aliquots that were plated on LB agar plates(16) . For measuring total cellular KDNase activity, the cells were sedimented by centrifugation at 5,000 rpm (1,500 times g) for 10 min, resuspended in 0.5 ml of 100 mM Tris acetate buffer, pH 6.0, containing 100 mM NaCl, and sonically disrupted (50 watts, 1 min). KDNase activity was assayed in the supernatant fraction obtained after centrifugation at 10,000 rpm (6,000 times g) for 10 min.

Release of KDNase SM by Osmotic Shock from S. multivorum

Washed cells (1.2 times 10), grown at 25 °C in 160 ml of M9 medium supplemented with 1% (w/v) casamino acids and glucose, as described above, were inoculated into 500 ml of M9 medium containing 0.1% (w/v) enriched KDN-OS as the sole carbon source, and incubated at 25 °C for 43 h. The induced cells were harvested, and the periplasmic fraction was prepared according to the cold osmotic shock procedure of Nossal and Heppel(22) . In brief, cells (8 g, wet weight) were harvested and washed in cold 0.03 M NaCl-0.01 M Tris-HCl buffer (pH 7.1) by centrifugation at 13,000 times g for 20 min. The washed cell pellet was weighed and resuspended in 10 volumes (v/w) of 0.033 M Tris-HCl buffer, pH 7.1, followed by the addition, with rapid stirring, of 10 vol (v/w) of 40% (w/v) sucrose in the same buffer that contained 10 vol (v/w) of 0.1 M Na(2)EDTA, pH 7.1. After incubation with shaking at room temperature for 10 min, the sucrose-treated cells were collected by centrifugation (13,000 times g/10 min), and resuspended in 20 vol (v/w) of ice-cold 1 mM Mg(CH(3)COO)(2) with gentle stirring for 10 min. After the addition of 2 vol (v/w) of ice cold 1 M NaCl, 1 M Tris-HCl buffer (pH 7.1), the suspension was centrifuged (13,000 times g/30 min). Enzymes localized in the periplasm were recovered in the supernatant(22) .

Purification of KDNase SM from the Periplasm of S. multivorum

KDNase SM was precipitated from the periplasmic fraction by the gradual addition of solid ammonium sulfate, with gentle stirring, to 90% saturation. After sitting overnight at 4 °C, the precipitate was collected by centrifugation at 17,000 times g for 30 min, dissolved in 10 ml of 0.1 M NaCl-100 mM Tris acetate, pH 6.0, and dialyzed against 0.1 M NaCl, 0.25 M sucrose, 20 mM Tris acetate, pH 6.0. The dialyzed solution was applied to a CM-Toyopearl 650 M column (2.2 times 11 cm, 42 ml; equilibrated with the same dialysis buffer) and eluted first with 60 ml of the same buffer, followed by elution with 400 ml of a linear NaCl gradient (0.1-0.6 M) in 0.25 M sucrose-20 mM Tris acetate, pH 6.0. The flow-through fractions were combined, concentrated to 15 ml by limited filtration (Amicon, YM10), and applied to a DEAE-Toyopearl 650 M column (2.2 times 11 cm, 42 ml), which had been equilibrated with 0.1 M NaCl, 0.25 M sucrose, 20 mM Tris-HCl buffer, pH 8.0. The column was eluted initially with 60 ml of the same buffer as used for the equilibration, and subsequently with 60 ml of 0.5 M NaCl, 0.25 M sucrose, 20 mM Tris-HCl buffer, pH 8.0. The unbound fractions were pooled, concentrated to 13 ml by limited filtration, and chromatographed on a CM-Toyopearl 650 M column, as described above. Six ml-fractions were collected and assayed for KDNase activity and protein, the latter by measuring absorbance at 280 nm.

Chemical Analysis

Protein was quantitated by the modified Lowry method (BCA, Pierce Chemical Co.) using bovine serum albumin as the standard.

Properties of KDNase SM

The apparent molecular weight of KDNase SM was estimated by gel filtration on Sephacryl S-200 and SDS-polyacrylamide gel electrophoresis, as described previously (16) .

The effect of pH on the KDNase SM-catalyzed hydrolysis of 4-MU-KDN was studied by incubating the enzyme (2 µl; 7.0 milliunits) with 4-MU-KDN (1.6 µl; 1.6 nmol) in 20 µl of 0.1 M NaCl-containing 0.1% (w/v) BSA and 0.1 M Tris acetate buffer that was adjusted to pH 4.5-8.0. The effects of sodium cholate and Triton X-100 on the activity were examined at concentrations up to 0.5% (w/v).

The effect of temperature on KDNase activity was tested by incubating the enzyme at 4, 15, 25, and 37 °C. Thermal stability of the enzyme was also estimated by measuring the activity after preincubation of the reaction mixture at 4, 25, and 37 °C for 0, 15, 30, and 60 min.


RESULTS

Growth Properties of Sphingobacterium multivorum and Induction of KDNase SM

The doubling time of S. multivorum in LB medium at 25 °C was approximately 50 min. A decrease in KDNase activity in both the periplasmic fraction and the sonically disrupted cell homogenate was observed when cells grown initially in the presence of KDN-oligosaccharide alditols were grown without inducer. For example, after five transfers of an induced culture in the absence of inducer, the KDNase activity decreased by 90% of that of the original level and then remained constant at about 1.2 unit/g wet cell weight. During this attenuation in KDNase activity, the doubling time and cell volumes appeared unchanged. However, when these cells were incubated with 0.1% KDN-OS as inducer in M9 minimal medium, KDNase activity was restored. As shown in Fig. 1and Table 1, KDNase activity per cell was 15-40-fold higher, compared with growth in the absence of inducer. On the basis of these results, we conclude that KDNase is an inducible enzyme in S. multivorum and that KDN-oligosaccharides appear to be required for induction. To confirm this finding, cells were grown for 24 h in M9 minimal medium containing the various mono- and oligosaccharides shown in Table 1, as sole carbon sources. KDN-OS and enriched KDN-OS were shown to be the only inducers of KDNase SM activity (Table 1). Free KDN had little effect on induction of KDNase SM, indicating that the ketosidic linkage of bound KDN residues was a structural requirement for induction. It is also noted in Table 1that the cell number remained essentially unchanged during the 24 h period when 0.1% KDN, Neu5Ac, or oligoNeu5Ac were the sole carbon sources. In contrast, KDN-OS, enriched KDN-OS, and glucose were effective carbon sources, although glucose was not an inducer.


Figure 1: Induction of KDNase SM in S. multivorum with enriched KDN-OS. Cells grown initially in M9 medium supplemented with 1% casamino acids and glucose were harvested and incubated in M9 minimal medium containing 0.1% KDN-OS as the sole carbon source. At the times indicated, KDNase activity and viable cell number were determined, as described under ``Experimental Procedures.''





KDNase SM Is a Periplasmic Enzyme in S. multivorum

Because a number of bacterial hydrolytic enzymes including acid and alkaline phosphatases, carboxypeptidases, polyphosphatases, sugar phosphate phosphohydrolyases, 5`-nucleotidase, and sugar hydrolylases are localized in the periplasmic space(23) , we sought to determine if KDNase SM was a periplasmic enzyme. Cold osmotic shock is a method commonly used to release periplasmic proteins(22) . As shown in Table 2, nearly all (99%) of the KDNase activity in the cell homogenate of induced cells of S. multivorum was released by osmotic shock. Importantly, the specific activity of the enzyme in the periplasm was 4.4-fold higher than in the cell homogenate (120 versus 27 units/mg of protein). As described below, we were able to exploit this finding and purify the enzyme after osmotic shock 700-fold to homogeneity.



Purification of KDNase SM from S. multivorum

KDNase SM was released by osmotic shock from the periplasm of induced cells of S. multivorum grown for 43 h in M9 medium containing 0.1% enriched KDN-OS and purified to homogeneity. The enzyme was first precipitated by 90% ammonium sulfate before being applied to a cation-exchange CM-Toyopearl 650 M column. The KDNase activity obtained in the flow-through fractions (1st CM-Toyopearl fraction) was applied to an anion-exchange DEAE-Toyopearl 650M column. The KDNase active fractions were not retained by this column, and the flow-through fractions (DEAE-Toyopearl fraction) were subjected to a second CM-Toyopearl 650M chromatography. As shown in Fig. 2, the KDNase activity now eluted at an NaCl concentration of 0.2-0.22 M (2nd CM-Toyopearl fraction). The KDNase activity was not adsorbed to the first CM-Toyopearl column, because the enzyme appears to have formed a complex with highly negatively charged molecules, possibly DNA or RNA, that were removed by the DEAE-Toyopearl column. The KDNase fractions at each purification step were analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 3), and the 2nd CM-Toyopearl fraction was shown to be pure, based on the fact that only a single band was observed when the gel was stained by the silver staining method (Fig. 3, lane 4). As summarized in Table 3, KDNase was purified 700-fold in 43% yield from the periplasmic fraction. The specific activity of the pure enzyme was 82100 unit/mg of protein. The KDNase SM thus obtained was highly specific for KDN ketosidic linkages of natural and synthetic substrates and, as reported for the partially purified enzyme, did not hydrolyze 4-MU-Neu5Ac, or other N-acylneuraminyl glycan chains(16) . No protease, other glycosidases, or peptide:N-glycanase activities were detected. The purified enzyme was also free of the low levels of alpha-fucosidase and alpha- and beta-hexosaminidase activities that were detected previously in the affinity-purified KDNase SM(16) .


Figure 2: Purification of KDNase SM from S. multivorum by CM-Toyopearl 650M chromatography. The flow-through fractions from the DEAE-Toyopearl chromatography step were applied to a column of CM-Toyopearl 650 M and eluted first with 0.25 M sucrose-20 mM Tris-acetate buffer, pH 6.0, containing 0.1 M NaCl, followed by elution with a linear NaCl gradient (0.1-0.6 M) in the same buffer. 6-ml fractions were collected and assayed for KDNase activity. The protein concentration in the KDNase active fractions is not shown because it was too low to be determined without significant concentration.




Figure 3: SDS-polyacrylamide gel electrophoresis of fractions obtained at each purification step. Molecular weights are indicated on the side. Lane 1, periplasmic fraction; lane 2, first CM-Toyopearl pooled fractions; lane 3, pooled DEAE-Toyopearl fractions; lane 4, second CM-Toyopearl fraction.





Determination of Apparent Molecular Weight

When the purified KDNase was chromatographed on an analytical Sephacryl S-200 column, a single peak of KDNase activity was obtained that eluted with an apparent molecular weight of 40,000. The apparent molecular weight of the enzyme was also estimated to be 47,500 by SDS-polyacrylamide gel electrophoresis (Fig. 3, lane 4), both in the presence and absence of 2.5% mercaptoethanol. This finding indicated that KDNase SM consisted of a single polypeptide chain with an apparent M(r) of approximately 47,500.

Instability

The purified KDNase SM was extremely unstable in the absence of added protein, and lost a considerable amount of activity. Hence, the enzyme was protected from denaturation by the addition of bovine serum albumin at 0.1-1.0 mg/ml to the incubation mixtures. KDNase activity remained unchanged on storage at 4 °C in the presence of 0.1% BSA for at least 6 months. As shown in Fig. 4, KDNase activity decreased by 90% after incubation at 37 °C for 15 min even in the presence of 0.1% BSA. The effect of detergents often used to increase the susceptibility of glycolipid substrates to glycosidases had variable effects on KDNase activity. Triton X-100 at concentrations of 0.1-0.5% (w/v) stimulated the activity by 10-20%. In contrast, 82% of the enzyme activity was inhibited by 0.5% (w/v) sodium cholate, while 90% of the activity was retained at 0.1%.


Figure 4: Thermal stability of KDNase SM. KDNase activity was determined after incubation at 4 °C (circle), 25 °C (bullet), 37 °C () for 15, 30, and 60 min in the presence of 0.1% BSA.



Effect of pH, Temperature, and Salt on KDNase SM Activity

The pH activity profile of KDNase SM was examined in the presence of 0.1% BSA. Activity was maximal in the pH range between 6.0 and 7.0, and at 25 °C. Activity decreased at 37 °C, because of the instability of the enzyme at this temperature (Fig. 4). No requirement for divalent cations could be demonstrated. Exposure of the enzyme to low concentrations of NaCl (less than 0.1 M) for 30 min reversibly inactivated the enzyme. Exposure for more than 30 min irreversibly inactivated the enzyme.


DISCUSSION

Our new findings show that KDNase SM in S. multivorum is an inducible enzyme that is localized in the periplasm. The specific activity in the periplasm was 9- and 4-fold higher than that in the sonic cell homogenate of the uninduced and induced cells, respectively: 6.4 versus 0.737 units/mg of protein for uninduced cells, 120 versus 27 units/mg protein for induced cells. After induction with KDN-OS or enriched KDN-OS, KDNase activity was increased 15-40-fold, and the specific activity in the periplasm was 117 units/mg of protein, which is 20-fold higher than before induction. These facts allowed us to release the enzyme from induced cells by osmotic shock, and to purify it 700-fold to homogeneity. A ketosidic linkage of KDN was necessary for induction, because neither free KDN, Neu5Ac, or oligoNeu5Ac was an inducer. Thus, a regulatory protein responsible for KDNase induction would presumably require a KDN ketosidic linkage, although the molecular mechanism for induction is not known.

KDNase SM consists of a single polypeptide chain having an estimated molecular weight of 47,500. It is a weakly basic protein, based on its retention on a cation-exchange column of CM-Toyopearl at pH 6.0. KDNase SM has a pH optimum at 6.0-7.0. The purified enzyme is extremely labile, but it can be stabilized by the addition of 0.1% BSA. Complete loss of the activity was observed after a 15-min incubation at 37 °C, but below 25 °C the enzyme lost only about 10% of its activity after 60 min. KDNase SM is active in the presence of 0.5% Triton X-100, conditions which facilitate the hydrolysis of KDN residues in KDN-gangliosides. The enzyme is highly specific for ketosidic linkages of KDN in oligosaccharides, glycoproteins, and glycolipids as well as a synthetic substrate, 4-MU-KDN, and devoid of any N-acylneuraminidase activities, as was previously shown for the partially purified enzyme(16) . These properties and substrate specificity indicate the usefulness of KDNase SM for studies that seek to elucidate the structure-function of KDN-glycoconjugates. In view of the recent success in cloning of a number of N-acylneuraminidases and in determining their tertiary structures(24) , it will be of particular interest to obtain KDNase SM in pure crystalline form for x-ray crystallographic analysis.


FOOTNOTES

*
This research was supported by Grant-in-aid for International Scientific Research Program: Joint Research 04044055 (to Y. I.) from the Ministry of Education, Science, and Culture of Japan and by Grants-in-aid for General Scientific Research 07680647 and for Developmental Scientific Research 07558211 (to K. K.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: KDN, 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid; Sia, sialic acid; KDN-glycoconjugates, KDN-containing glycoconjugates; KDNase, an enzyme which catalyzes the hydrolysis of ketosidic linkages of KDN; KDN-OS, KDNalpha2-3Galbeta1-3GalNAcalpha1-3[KDNalpha2-(8KDNalpha2-)-6]GalNAcol with n = 5; 4-MU-KDN, 4-methylumbelliferyl KDN; KDNase SM, KDNase isolated from Sphingobacterium multivorum; BSA, bovine serum albumin.

(^2)
M. Ziak, B. Qu, X. Zuo, C. Zuber, A. Kanamori, K. Kitajima, S. Inoue, Y. Inoue, and J. Roth, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Thomas G. Warner, Genentech, Inc., for providing 4-methylumbelliferyl KDN.


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