Substrate specificity of rat brain ceramidase.

In this study, the substrate specificity of a newly identified rat brain ceramidase (CDase) was investigated. To this end, the major functional groups and stereochemistry of ceramide (Cer) were evaluated for their influence on the hydrolysis of substrate by this CDase. The results showed that, of the four possible stereoisomers of Cer, only the natural d-e-C(18)-Cer isomer was used as substrate (K(m) of 1.1 mol% and V(max) of 5 micromol/min/mg). Removal of the 4-5 trans double bond to generate dihydroceramide decreased the affinity of the enzyme toward its substrate by around 90%, whereas changing the configuration of the double bond from the natural trans configuration into cis or introduction of a hydroxyl group (phytoceramide) resulted in loss of hydrolysis. Shortening the chain length of the sphingosine backbone resulted in decreased affinity. Methylation of either the primary or the secondary hydroxyl groups resulted in loss of activity. Results also indicated that Cer species that harbor long saturated or monounsaturated fatty acyl chains are preferred substrates of the enzyme. alpha-Hydroxylated Cer demonstrated considerably higher affinity, indicating a preference of the enzyme to those Cer molecular species. These results disclose a very high specificity of nonlysosomal CDase for its substrate, Cer.     

Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

We report here the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue (AE003774) was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product.     

Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain.

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C(16)-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K(m) of 1.29 mol % and a V(max) of 4.4 micromol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 micromol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC(50) of 5-10 microM. These results indicate that the purified enzyme is a novel ceramidase.     

Characterization of a neutral ceramidase orthologue from Aspergillus oryzae

Ceramide is an important molecule not only structurally but also regulationally as a modulator of various cellular events. Ceramidase (CDase) are classified into three different types (acid, alkaline, and neutral CDases). Neutral CDase could play an important role in the regulation of ceramide levels in the extracellular space. In this study, we describe the characterization of a neutral CDase orthologue from the filamentous fungus Aspergillus oryzae . The gene encoding the neutral CDase orthologue was cloned and overexpressed in A . oryzae . The purified recombinant enzyme was optimally active at pH 4.0–4.5 and 40 °C. The apparent K _m and V _max values of the enzyme for C12-NBD-ceramide were 3.32 μM and 0.085 μmol min⁻¹ mg⁻¹, respectively.     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110kDa has an optimal pH of 7.0 and a pI of 5.6. It splits beta-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl beta-naphthylamide (Leu betaNA) is the best substrate with the highest hydrolytic coefficiency followed by Met betaNA=Arg betaNA=Lys betaNA>Ala betaNA>Tyr betaNA>Phe betaNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K(m) 82microM and a k(cat) of 1.08s(-1), and Met-enkephalin with a K(m) of 106microM and a k(cat) of 2.6s(-1). The puromycin-sensitive enzyme is most susceptible to amastatin with an IC(50) of 0.05microM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

New insight into the structure,reaction mechanism,and biological functions of neutral ceramidase

Ceramidase (CDase) is an enzyme that hydrolyzes the N-acyl linkage between the sphingoid base and fatty acid of ceramide. These enzymes are classified into three distinct groups, acid (Asah1), neutral (Asah2), and alkaline (Asah3) CDases, based on their primary structure and optimum pH. Acid CDase catabolizes ceramide in lysosomes and is found only in vertebrates. In contrast, the distribution of neutral and alkaline CDases is broad, with both being found in species ranging from lower eukaryotes to mammals; however, only neutral CDase is found in prokaryotes, including some pathogenic bacteria. Neutral CDase is thought to have gained a specific domain (mucin box) in the N-terminal region after the vertebrate split, allowing the enzyme to be stably expressed at the plasma membrane as a type II membrane protein. The X-ray crystal structure of neutral CDase was recently solved, uncovering a unique structure and reaction mechanism for the enzyme. Neutral CDase contains a zinc ion in the active site that functions as a catalytic center, and the hydrolysis of the N-acyl linkage in ceramide proceeds through a mechanism that is similar to that described for zinc-dependent carboxypeptidase. This review describes the structure, reaction mechanism, and biological functions of neutral CDase in association with the molecular evolution, topology, and mechanical conformation. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain.

Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein.     

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 microg of extracellular alkaline, Ca(2+)-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5alpha and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5alpha culture, 280 microg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8 mg of pure active CDase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield CDase production system allowing detailed characterization of the enzyme at the molecular level.     

Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain

Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Purification and properties of rat brain dipeptidyl aminopeptidase

Dipeptidyl aminopeptidase, which hydrolyzes the 7-(Gly-Pro)-4-methylcoumarinamide, has been purified from the brains of 3 week-old rats. It was purified about 2,600-fold by column chromatography on CM-cellulose, hydroxyapatite and Gly-Pro AH-Sepharose. This enzyme hydrolyzed Lys-Ala-beta-naphthylamide well with an optimum pH of 5.5. It was inhibited by diisopropyl fluorophosphate, phenyl-methanesulfonyl fluoride, some cations, and puromycin, but was not inhibited by p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, EDTA, iodoacetic acid, and bacitracin, indicating that rat brain dipeptidyl aminopeptidase is a serine protease. This enzyme showed a molecular weight of 220,000 by gel filtration and of 51,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The properties of purified rat brain dipeptidyl aminopeptidase were similar to those of bovine pituitary dipeptidyl peptidase II, but the molecular weight and substrate specificity of these enzymes were different.     

Purification of a novel type of calcium-activated neutral protease from rat brain. Possible involvement in production of the neuropeptide kyotorphin from calpastatin fragments

We have found a novel type of Ca2(+)-activated neutral protease in rat brain cytosol which cleaves -Tyr-Arg-containing calpastatin fragments to release the neuropeptide kyotorphin. This enzyme was purified about 26,000-fold by column chromatography as follows: DE52 cellulose, Ultrogel AcA 44, thiopropyl-Sepharose 6B, second DE52 cellulose, Ultrogel AcA 34, and blue Sepharose CL-6B. The molecular mass of the enzyme was estimated to be 65-75 kDa by gel filtration. The purified enzyme gave a single band of 74 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some properties of this enzyme were similar to those of the calpains, i.e. an absolute requirement for Ca2+, maximal activity at neutral pH, and inhibition by sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and N-ethylmaleimide. However, it differs from the calpains in that it possesses no caseinolytic activity, separates from the calpains on the first DE52 column, and is insensitive to leupeptin and E-64 (N-[N-(L-3-trans-carboxyoxrian-2-carbonyl)-L-leucyl]agmatine). Thus, the molecular mass, the substrate specificity, the chromatographic behavior, and the inhibitor spectrum all suggest that this enzyme is a novel type of Ca2(+)-activated neutral protease.     

Characterization of a novel alpha-D-mannosidase from rat brain microsomes

A new alpha-D-mannosidase has been identified in rat brain microsomes. The enzyme was purified 70-100-fold over the microsomal fraction by solubilization with Triton X-100, followed by ion exchange, concanavalin A-Sepharose, and hydroxylapatite chromatography. The purified enzyme is very active towards mannose-containing oligosaccharides and has a pH optimum of 6.0. Unlike rat liver endoplasmic reticulum alpha-D-mannosidase and both Golgi mannosidases IA and IB, which have substantial activity only towards alpha 1,2-linked mannosyl residues, the brain enzyme readily cleaves alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannosyl residues present in high mannose oligosaccharides. The brain enzyme is also different from liver Golgi mannosidase II in that it hydrolyzes (Man)5GlcNAc and (Man)4GlcNAc without their prior N-acetylglucosaminylation. Moreover, the facts that the ability of the enzyme to cleave GlcNAc(Man)5GlcNAc, the biological substrate for Golgi mannosidase II, is not inhibited by swainsonine, and that p-nitrophenyl alpha-D-mannoside is a poor substrate provide further evidence for major differences between the brain enzyme and mannosidase II. Inactivation studies and the co-purification of activities towards various substrates suggest that a single enzyme is responsible for all the activities found. In view of these results, it seems possible that, in rat brain, a single mannosidase cleaves asparagine-linked high mannose oligosaccharide to form the core Man3GlcNAc2 moiety, which would then be modified by various glycosyl transferases to form complex type glycoproteins.     

A comparison of the properties of 5'-nucleotidase purified from the cytosolic and synaptic plasma membrane fractions of rat forebrain.

1. 5'-Nucleotidase was purified 1247-fold from the post-microsomal supernatant (I) and 3862-fold from the synaptic plasma membrane (II) of rat brain homogenates. 2. The apparent molecular masses of I and II were 131 and 72 kDa respectively by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and 268 and 286 kDa respectively by Sephacryl S-300 chromatography. 3. The activities of both I and II were strongly inhibited by concanavalin A but were affected differently by digestion with glycosidases. for II, these were 0.083 and 0.056 mM respectively. 5. Activities of both I and II were strongly inhibited by ATP and ADP.     

Isolation of similar rolipram-inhibitable cyclic-AMP-specific phosphodiesterases from rat brain and heart

A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 microM, while hydrolysis of cyclic GMP displayed anomalous negatively cooperative kinetics. At micromolar concentrations, this enzyme from hydrolyzed highly specifically cyclic AMP (50-fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 microM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties, probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipram-sensitive form between the two tissues. Since the rolipram-sensitive form detected in crude brain preparations markedly differs from the above-described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.     

Molecular cloning and characterization of a human mitochondrial ceramidase

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism.     

Purification and characterization of human intestinal neutral ceramidase

Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu(2+) and Zn(2+) ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.     

Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.     

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn²⁺. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn²⁺-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC₅₀ of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn²⁺.Abbreviations PP1-C catalytic subunit of type 1 protein phosphatase - SDS sodium dodecyl sulfate - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - Mops 4-morpholine propanesulfonic acid - EDTA ethylenediaminetetraacetate - EGTA [ethylenbis (oxyethylenenitrilo)]-tetra acetic acid     

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain

Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.