Human anionic trypsinogen: properties of autocatalytic activation and degradation and implications in pancreatic diseases.

Human pancreatic secretions contain two major trypsinogen isoforms, cationic and anionic trypsinogen, normally at a ratio of 2 : 1. Pancreatitis, pancreatic cancer and chronic alcoholism lead to a characteristic reversal of the isoform ratio, and anionic trypsinogen becomes the predominant zymogen secreted. To understand the biochemical consequences of these alterations, we recombinantly expressed and purified both human trypsinogens and documented characteristics of autoactivation, autocatalytic degradation and Ca2+-dependence. Even though the two trypsinogens are approximately 90% identical in their primary structure, we found that human anionic trypsinogen and trypsin exhibited a significantly increased (10-20-fold) propensity for autocatalytic degradation, relative to cationic trypsinogen and trypsin. Furthermore, in contrast to the characteristic stimulation of the cationic proenzyme, acidic pH inhibited autoactivation of anionic trypsinogen. In mixtures of cationic and anionic trypsinogen, an increase in the proportion of the anionic proenzyme had no significant effect on the levels of trypsin generated by autoactivation or by enterokinase at pH 8.0 in 1 mm Ca2+- conditions that were characteristic of the pancreatic juice. In contrast, rates of trypsinogen activation were markedly reduced with increasing ratios of anionic trypsinogen under conditions that were typical of potential sites of pathological intra-acinar trypsinogen activation. Thus, at low Ca2+ concentrations at pH 8.0, selective degradation of anionic trypsinogen and trypsin caused diminished trypsin production; while at pH 5.0, inhibition of anionic trypsinogen activation resulted in lower trypsin yields. Taken together, the observations indicate that up-regulation of anionic trypsinogen in pancreatic diseases does not affect physiological trypsinogen activation, but significantly limits trypsin generation under potential pathological conditions.     

The two human trypsinogens. Evidence of complex formation with basic pancreatic trypsin inhibitor-proteolytic activity.

The formation of complexes between human trypsinogens and the basic pancreatic trypsin inhibitor is demonstrated by using affinity chromatography on Sepharose coupled to basic pancreatic trypsin inhibitor. This interaction indicates the pre-existence of the active site in human trypsinogens. This active site induces the proteolytic activity of the two zymogens which activate spontaneously at pH 5.6 and pH 8.0 before and after affinity chromatography. The effect of affinity-chromatography on trypsinogen spontaneous activation is not the same on trypsinogens 1 and 2. A striking difference appears between the activation of the two trypsinogens. In all cases, trypsinogen 1 autoactivates more rapidly than trypsinogen 2, except at pH 5.6 in the presence of 10 mM Ca2+, which inhibits the autoactivation of trypsinogen 1. The effect of inherent proteolytic activity of human trypsinogens is discussed in relation to pathological conditions of enterokinase deficiency and acute pancreatitis.     

Human cationic trypsinogen is sulfated on Tyr154

The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy-Crotte O, Forest E & Fontecilla-Camps JC (1996) J Mol Biol259, 995-1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation-exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [(35)S]SO(4) into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated pancreatic cationic trypsinogen exhibited faster autoactivation than a nonsulfated recombinant form, suggesting that tyrosine sulfation of trypsinogens might enhance intestinal digestive zymogen activation in humans. Finally, sequence alignment revealed that the sulfation motif is only conserved in primate trypsinogens, suggesting that typsinogen sulfation is absent in other vertebrates.     

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin,reveals its resistance to proteinaceous trypsin inhibitors

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.     

Human cationic trypsinogen. Role of Asn-21 in zymogen activation and implications in hereditary pancreatitis

Mutation Asn-21 --> Ile in human cationic trypsinogen (Tg-1) has been associated with hereditary pancreatitis. Recent studies with rat anionic Tg (Tg-2) indicated that the analogous Thr-21 --> Ile mutation stabilizes the zymogen against autoactivation, whereas it has no effect on catalytic properties or autolytic stability of trypsin (Sahin-Tóth, M. (1999) J. Biol. Chem. 274, 29699-29704). In the present paper, human cationic Tg (Asn-21-Tg) and mutants Asn-21 --> Ile (Ile-21-Tg) and Asn-21 --> Thr (Thr-21-Tg) were expressed in Escherichia coli, and zymogen activation, zymogen degradation, and trypsin autolysis were studied. Enterokinase activated Asn-21-Tg approximately 2-fold better than Ile-21-Tg or Thr-21-Tg, and catalytic parameters of trypsins were comparable. At 37 degrees C, in 5 mm Ca(2+), all three trypsins were highly stable. In the absence of Ca(2+), Asn-21- and Ile-21-trypsins suffered autolysis in an indistinguishable manner, whereas Thr-21-trypsin exhibited significantly increased stability. In sharp contrast to observations with the rat proenzyme, at pH 8.0, 37 degrees C, autoactivation kinetics of Asn-21-Tg and Ile-21-Tg were identical; however, at pH 5. 0, Ile-21-Tg autoactivated at an enhanced rate relative to Asn-21-Tg. Remarkably, at both pH values, Thr-21-Tg showed markedly higher autoactivation rates than the two other zymogens. Finally, autocatalytic proteolysis of human zymogens was limited to cleavage at Arg-117, and no digestion at Lys-188 was detected. The observations indicate that zymogen stabilization by Ile-21 as observed in rat Tg-2 is not characteristic of human Tg-1. Instead, an increased propensity to autoactivation under acidic conditions might be relevant to the pathomechanism of the Asn-21 --> Ile mutation in hereditary pancreatitis. In the same context, faster autoactivation and increased trypsin stability caused by the Asn-21 --> Thr mutation in human Tg-1 might provide a rationale for the evolutionary divergence from Thr-21 found in other mammalian trypsinogens.     

Purification and N-terminal amino acid sequence determination of anionic and cationic canine trypsinogens.

Anionic and cationic canine trypsinogens were purified from pancreatic juice by affinity chromatography with Trasylol coupled to Sepharose 4B followed by ion exchange chromatography with SP-Sephadex C-50. Automatic N-terminal amino acid sequence determination showed the following structures for the activation peptides: Thr-Pro-Thr-Asp-Asp-Asp-Asp-Lys for anionic trypsinogen, and Phe-Pro-Ile-Asp-Asp-Asp-Asp-Lys for cationic trypsinogen.     

Further studies of an inactive form of a trypsin-like enzyme in rabbit testes

An inactive form of rabbit testicular trypsin-like activity was partially purified utilizing acid pH conditions for extraction, dialysis, and Sephadex gel filtration and ion-exchange chromatography. An inhibitor of the testicular trypsin-like activity was separated from the inactive form, but the latter still remained inactive until incubation with bovine pancreatic trypsin or “autoactivation” at pH 8. “Autoactivation” followed a sigmoidal time course and was not reversible. These results suggest the existence of a zymogen form of rabbit testicular trypsin-like activity.     

Hereditary pancreatitis-associated mutation asn(21) --> ile stabilizes rat trypsinogen in vitro.

Mutations Arg(117) --> His and Asn(21) --> Ile in human trypsinogen-I have been recently associated with hereditary pancreatitis (HP). The Arg(117) --> His substitution is believed to cause pancreatitis by stabilizing trypsin against autolytic degradation, while the mechanism of action of Asn(21) --> Ile has been unknown. In an effort to understand the effect(s) of this mutation, Thr(21) in the highly homologous rat trypsinogen-II was replaced with Asn or Ile, and the recombinant zymogens and their active trypsin forms were studied. Kinetic parameters of all three trypsins were comparable, and the active enzymes suffered autolysis at similar rates, indicating that neither catalytic properties nor proteolytic stability of trypsin are influenced by mutations at position 21. When incubated at pH 8.0, 37 degrees C, pure zymogens underwent autoactivation with concomitant trypsinolytic degradation in a Ca(2+)-dependent fashion. Thus, in the presence of 5 mM Ca(2+), autoactivation and digestion of the zymogens after Arg(117) and Lys(188) were observed, while in the presence of 1 mM EDTA autoactivation and cleavage at Lys(188) were reduced, and zymogenolysis at the Arg(117) site was enhanced. Overall rates of zymogen degradation in [Asn(21)]- and [Ile(21)]trypsinogens were higher in Ca(2+) than in EDTA, while [Thr(21)]trypsinogen demonstrated inverse characteristics. Remarkably, both in the presence and absence of Ca(2+), [Ile(21)]trypsinogen exhibited significantly higher stability against autoactivation and proteolysis than zymogens with Asn(21) or Thr(21). The observations suggest that autocatalytic trypsinogen degradation may be an important defense mechanism against excessive trypsin generation in the pancreas, and trypsinogen stabilization by the Asn(21) --> Ile mutation plays a role in the pathogenesis of HP.     

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.     

Limited proteolyses in pancreatic chymotrypsinogens and trypsinogens

In the 1950's, the specific cleavages of the peptide bonds occurring in bovine cationic chymotrypsinogen and trypsinogen were among the first examples of limited proteolyses. According to the split bond(s), the precursor is transformed into enzyme or different forms of zymogen or again into inert protein. The conversion of trypsinogen into trypsin triggers the activations of all the other enzyme precursors of pancreatic juice. In the pancreas, several factors oppose trypsinogen autoactivation, whereas, in the duodenum, all the conditions are favorable for trypsinogen activation by enteropeptidase.     

Comparative studies on the mechanism of activation of the two human trypsinogens

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.     

Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand

An ammonium sulphate fraction (20–60%) of bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) was purified by affinity chromatography to give 6.59-fold purity with 81.48% yield. The same ammonium sulphate fraction was also subjected to ion exchange chromatography and was purified 4.28-fold with 75.95% yield. The ion exchange fraction was subjected to gel filtration and the inhibitor was purified to 6.67-fold with 67.36% yield. Further sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the homogeneity of purified amylase/trypsin inhibitor obtained through affinity, ion exchange and gel chromatography. The molecular weight of the inhibitor was found to be 14 kDa. This purified inhibitor was used as affinity ligand for the purification of a commercial preparation of pancreatic amylase.     

Trypsinogen stabilization by mutation Arg117-->His: a unifying pathomechanism for hereditary pancreatitis?

Mutations Arg117-->His and Asn21-->Ile of the human cationic trypsinogen have been recently identified in patients affected by hereditary pancreatitis (HP). The Arg117-->His substitution is believed to cause pancreatitis by eliminating an essential autolytic cleavage site in trypsin, thereby rendering the protease resistant to inactivation through autolysis. Here we demonstrate that the Arg117-->His mutation also significantly inhibits autocatalytic trypsinogen breakdown under Ca(2+)-free conditions and stabilizes the zymogen form of rat trypsin. Taken together with recent findings demonstrating that the Asn21-->Ile mutation stabilizes rat trypsinogen against autoactivation and consequent autocatalytic degradation, the observations suggest a unifying molecular pathomechanism for HP in which zymogen stabilization plays a central role.     

Phospholipase D from savoy cabbage: purification and preliminary kinetic characterization

Phospholipase D has been purified 680-fold from an acetone powder of savoy cabbage in an overall yield of 30%. The purification involves solubilization of the acetone powder in a Ca2+-containing buffer and subsequent ammonium sulfate fractionation. Gel filtration on Sephadex G-200 and hydrophobic affinity chromatography using a gamma-aminopropane-agarose gel complete the purification. The two chromatographic steps were conducted in buffers containing 50% ethylene glycol, which was necessary in order to maintain stability of the enzyme. Purity was established on the basis of gel electrophoresis and ultracentrifugation. A preliminary kinetic characterization of the enzyme was carried out by using lecithins with short-chain fatty acids below the critical micelle concentration. A complex series of results were obtained which demonstrated the following. (1) The enzyme is quite sensitive to ionic strength, being inhibited at high ionic strength. (2) The pH optimum depends on the concentration of Ca2+ used in the assay. At 0.5 mM Ca2+ the pH optimum is 7.25, but it is 6.0 at 50 mM Ca2+. (3) The effect of substrate concentration at a given pH and ionic strength did not show simple hyperbolic kinetics but rather regions of parabolic and hyperbolic kinetics.     

Increased activation of hereditary pancreatitis-associated human cationic trypsinogen mutants in presence of chymotrypsin C

Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.     

Proteolysis of human trypsinogen 1. Pathogenic implication in chronic pancreatitis

SDS electrophoresis on polyacrylamide gels of purified trypsinogen 1 has shown the occurence of a proteolysis in some molecules during long storage at ?20°C. This proteolyzed trypsinogen gives a positive reaction with an antiserum directed against the precipitate protein, major protein of about 14 000 molecular weight extracted from precipitates present in the pancreatic juice of patients with chronic pancreatitis. The autoactivation of proteolyzed trypsinogen 1 liberates a polypeptide of 14 000 molecular weight which is immunologically identical to the precipitate protein. These results show that the major protein present in pancreatic precipitates (and pancreatic stones) of patients with chronic pancreatitis is a degradation product of trypsinogen 1 liberated by a proteolysis which necessarily requires a premature zymogen activation in the disease.     

High-affinity Ca(2+) binding inhibits autoactivation of rat trypsinogen

The recent discovery that mutation Asn21 --> Ile in the human cationic trypsinogen (Tg) is associated with hereditary pancreatitis has brought into focus the functional role of amino acid 21 in mammalian Tgs. In the present paper, the effect of mutations Thr21 --> Asn and Thr21 --> Ile on the Ca(2+) dependence of zymogen activation was investigated, using the autolysis-resistant rat Tg mutant Arg117 --> His. In the absence of Ca(2+), rat Tg exhibited low but significant basal autoactivation, which was inhibited by micromolar concentrations of Ca(2+) (IC(50) 2.6 microM). Interestingly, basal autoactivation was diminished in both mutants, and no further inhibition by micromolar Ca(2+) was detectable. Millimolar Ca(2+) concentrations markedly and comparably stimulated autoactivation of wild-type and mutant zymogens (EC(50) 1.7-2.4 mM). The results indicate that rat Tg is subject to dual regulation by Ca(2+), allowing zymogen stabilization in a low-Ca(2+) environment and efficient activation in a high-Ca(2+) milieu.     

Partial purification and characterization of human sperminogen

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.     

Purification and properties of sulfhydryl oxidase from bovine pancreas

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.     

Hydrophobic-ionic chromatography. Its application to purification of porcine pancreas enzymes.

1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 AND 5.3) WITH 0.5 M acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 M 3,3-dimethyl glutarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of the pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fmately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases. The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.07 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.