Alkaline pH Optimum Research Articles

Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning, expression, purification, and characterization

Choline kinase is the first enzymatic step in the CDP–choline pathway for phosphatidylcholine biosynthesis. The genome of the nematode, Caenorhabditis elegans, contains seven genes that appear likely to encode choline and/or ethanolamine kinases. We cloned five and expressed four of these genes, and purified or partially purified three of the encoded enzymes. All expressed proteins had choline kinase activity; those that most closely resemble the mammalian choline kinases were the most active. CKA-2, a very active form, was purified to near homogeneity. The K m values for CKA-2 were 1.6 and 2.4 mM for choline and ATP, respectively, and k cat was 74 s −1. CKA-2 was predominantly a homodimer as assessed by glycerol gradient sedimentation and dynamic light scattering. CKB-2, which was less similar to mammalian choline kinases, had K m values for choline and ATP of 13 and 0.7 mM, and k cat was 3.8 s −1. Both of these highly purified enzymes required magnesium, had very alkaline pH optima, and were much more active with choline as substrate than with ethanolamine. These results provide a foundation for future studies on the structure and function of choline kinases, as well as studies on the genetic analysis of the function of the multiple isoforms in this organism.

Structure and function of a multidomain alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1

Xylanase is an enzyme that catalyzes the hydrolysis of xylan, a β-1,4-linked xylose polymer. Alkaliphilic Bacillus sp. strain 41M-1 secretes a xylanase (xylanase J) that has an alkaline pH optimum. Xylanase J is a multidomain enzyme and consists of two functional domains: a family 11/G catalytic domain and a non-catalytic xylan-binding domain. The xylan-binding domain bound to xylan and enhanced catalytic activity of the adjacent catalytic domain. Mutational analyses revealed some amino acid residues that contribute to catalytic activity, alkaliphily and xylan-binding activity of xylanase J.

Mouse brain serine racemase catalyzes specific elimination of L-serine to pyruvate

D-Serine was previously identified in mammalian brain and was shown to be a co-agonist at the ‘glycine’ site of the N-methyl-D-aspartate (NMDA)-type receptors. Racemization of serine is catalyzed by serine racemase, a pyridoxal 5′-phosphate-dependent enzyme expressed mainly in brain and liver. NMDA receptor overactivation has been implicated in a number of pathological conditions and inhibitors of serine racemase are thus potentially interesting targets for therapy. We expressed recombinant mouse serine racemase in insect cells and purified it to near homogeneity. The enzyme is a non-covalent homodimer in solution and requires divalent cations Mg2+, Ca2+ or Mn2+ for activity but not for dimerization. In addition to the racemization it also catalyzes specific elimination of L-Ser to pyruvate. D-Serine is eliminated much less efficiently. Both L-serine racemization and elimination activities of serine racemase are of comparable magnitude, display alkaline pH optimum and are negligible below pH 6.5.

Expression of Recombinant Plasmodium falciparumSubtilisin-like Protease-1 in Insect Cells: CHARACTERIZATION, COMPARISON WITH THE PARASITE PROTEASE, AND HOMOLOGY MODELING

Serine proteases play crucial roles in erythrocyte invasion by merozoites of the malaria parasite. Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is synthesized during maturation of the intraerythrocytic parasite and accumulates in a set of merozoite secretory organelles, suggesting that it may play a role in host cell invasion or post-invasion events. We describe the production, purification, and characterization of recombinant PfSUB-1 and comparison with the authentic protease detectable in parasite extracts. The recombinant protease requires high levels of calcium for optimum activity and has an alkaline pH optimum. Using a series of decapeptide and protein substrates, PfSUB-1 was found to have a relaxed substrate specificity with regard to the P1 position but is unable to efficiently cleave substrates with a P1 leucine residue. Similarly, replacement of a P4 valine with alanine severely reduced cleavage efficiency, whereas its replacement with lysine abolished cleavage. In all respects investigated, the recombinant protease was indistinguishable from parasite-derived enzyme. Three-dimensional homology modeling of the PfSUB-1 catalytic domain based on an alignment with closely related bacterial subtilisins and an orthologue from the rodent malaria Plasmodium yoelii suggests that the protease has at least three potential calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine within the enzyme S1 pocket. A predicted highly polar S1 pocket and a hydrophobic S4 subsite are in broad agreement with the experimentally determined substrate specificity.

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae).

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.

The Gene e.1 (nudE.1) of T4 Bacteriophage Designates a New Member of the Nudix Hydrolase Superfamily Active on Flavin Adenine Dinucleotide, Adenosine 5′-Triphospho-5′-adenosine, and ADP-ribose

The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5'-triphospho-5'-adenosine (Ap(3)A), and ADP-ribose. Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg(2+) or Mn(2+). For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap(3)A, the T4 enzyme hydrolyzes higher homologues including Ap(4-6)A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. Although the protein was predicted to be orthologous to E. coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following. (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates. (b) The e.1 gene does not complement mutT1 in vivo. (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage. The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue.

Activity of carbohydrases in the gut of Bibionidae (Diptera) larvae

Dipteran larvae play an important role in the soil of some deciduous forests. They can consume a considerable part of the annual litter fall and produce a large amount of faecal pellets, which are forming an important part of the fermentation horizon of forest soils. We measured the pH changes and assayed the activities and pH optima of saccharolytic enzymes in the digestive tracts of the larvae of Bibio pomonae and Penthetria holosericea (Bibionidae) . The pH of the litter offered as a food was about 5. The gut content became highly alkaline (pH about 10) in the anterior part of the midgut and the pH decreased posteriorly and in the hindgut. Excrements were neutral or had slightly alkaline pH (7.5–8.0) in both species. The alkaline pH optima of amylase (9.5–10.0) and maltase (8.0), trehalase and cellobiase (7.0) were the same in both species. Saccharolytic activity showed an optimum at pH 7 in B. pomonae and at pH 8 in P. holosericea. Though there are previously published reports of high assimilation efficiency of bibionid larvae fed in litter (46–76% in B. pomonae), activities of both exo and endocellulases were below the detection limit of saccharolytic and chromolytic assays along a wide pH range (5–10). Possible explanations are discussed.

Characterization of a novel insect digestive DNase with a highly alkaline pH optimum

A novel DNase from the digestive tract of the spruce budworm ( Choristoneura fumiferana) has been isolated and characterized. This DNase has two features that distinguish it from other known DNases: (1) it has a pH optimum of 10.5 to 11; (2) it plays an important role in the conversion of the insecticidal crystal protein from Bacillus thuringiensis to the active DNA-free toxin in the larval gut. Only one digestive DNase with an apparent molecular mass of 23 kDa was found and no associated carbohydrate was detected. It has some similarities to pancreatic DNase I in that divalent alkaline metal ion is required for activity and it is inhibited by monovalent cations. In particular, Mg 2+ and Ca 2+ were the most effective activators. Transition metal ions also activated the enzyme but were less effective. The enzyme is an endonuclease that hydrolyzes single and double stranded DNA but shows a higher specificity for single stranded DNA. The purified enzyme acted synergistically with proteases on crystals from Bacillus thuringiensis to yield the DNA-free toxin. To our knowledge, this is the first characterization of DNase activity in insect larvae and provides strong evidence that a DNase is an integral component of the larval digestive system.

Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris

Based on substrate specificity, an alkaline pH optimum, sensitivity to selected proteinase inhibitors, and molecular analysis, we provide evidence for the presence of a trypsin-like serine proteinase in the salivary gland complex (SGC) of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The predominant activity in extracts of the SGC against N 2-benzoyl- l-arginine- p-nitroanilide (L-BA pNA) was at pH 10, but a minor peak of activity also occurred at pH 5. The major BA pNAase activity focused at 10.4 during preparative isoelectric focusing and was eluted with an apparent molecular weight of 23,000 from a calibrated gel filtration column. The BA pNAase fraction gave a single major band when analyzed on a casein zymogram. The activity was completely suppressed by the serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor. A cDNA coding for a trypsin-like protein in the salivary glands of L. lineolaris was cloned and sequenced. The 971bp cDNA contained an 873-nucleotide open reading frame encoding a 291-amino acid trypsin precursor. The encoded protein included amino acid sequence motifs that are conserved with four homologous serine proteases from other insects. Typical features of the putative trypsin-like protein from L. lineolaris included the serine protease active site (His 89, Asp 139, Ser 229), conserved cysteine residues for disulfide bridges, the residues (Asp 223, Gly 252, Gly 262) that determine trypsin specificity, and both zymogen signal and activation peptides. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for trypsin like enzymes in the salivary glands of L. lineolaris.

Mutational Analysis of Baculovirus Capping Enzyme Lef4 Delineates an Autonomous Triphosphatase Domain and Structural Determinants of Divalent Cation Specificity

The 464-amino acid baculovirus Lef4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The hydrolysis of 5'-triphosphate RNA and free NTPs by Lef4 is dependent on a divalent cation cofactor. RNA triphosphatase activity is optimal at pH 7.5 with either magnesium or manganese, yet NTP hydrolysis at neutral pH is activated only by manganese or cobalt. Here we show that Lef4 possesses an intrinsic magnesium-dependent ATPase with a distinctive alkaline pH optimum and a high K(m) for ATP (4 mm). Lef4 contains two conserved sequences, motif A ((8)IEKEISY(14)) and motif C ((180)LEYEF(184)), which define the fungal/viral/protozoal family of metal-dependent RNA triphosphatases. We find by mutational analysis that Glu(9), Glu(11), Glu(181), and Glu(183) are essential for phosphohydrolase chemistry and likely comprise the metal-binding site of Lef4. Conservative mutations E9D and E183D abrogate the magnesium-dependent triphosphatase activities of Lef4 and transform it into a strictly manganese-dependent RNA triphosphatase. Limited proteolysis of Lef4 and ensuing COOH-terminal deletion analysis revealed that the NH(2)-terminal 236-amino acid segment of Lef4 constitutes an autonomous triphosphatase catalytic domain.

The Gene ygdP, Associated with the Invasiveness ofEscherichia coli K1, Designates a Nudix Hydrolase, Orf176, Active on Adenosine (5′)-Pentaphospho-(5′)-adenosine (Ap5A)

ygdP, a gene associated with the invasion of brain microvascular endothelial cells by Escherichia coli K1 (Badger, J. L., Wass, C. A., and Kim, K. S. (2000) Mol. Microbiol. 36, 174-182), the primary Gram-negative bacterium causing meningitis in newborns, has been cloned and expressed in E. coli. The protein, YgdP, was purified to near homogeneity and identified as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. It catalyzes the hydrolysis of diadenosine tetra-, penta-, and hexa-phosphates with a preference for diadenosine penta-phosphate, from which it forms ATP and ADP. The enzyme has a requirement for a divalent metal cation that can be met with Mg2+, Zn2+, or Mn2+ and, like most of the Nudix hydrolases, has an alkaline pH optimum between 8.5 and 9. This is the second identification of a gene associated with the invasiveness of a human pathogen as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases, and an examination of homologous proteins in other invasive bacteria suggests that this may be a common feature of cellular invasion.

Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant

The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala224) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Å (= 0.1nm) closer to the acid/base Glu217 residue, with a 3.1 Å contact between N∊2 and O∊1. The pH variation of kcat/Km for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK1 shifting from 2.22±0.03 in the wild-type to 3.19±0.03 in the mutant, and pK2 shifting from 5.99±0.02 to 6.78±0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative kcat for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower kcat/Km values for both lactosides and cellobiosides, and a marginally lower stability. However, kcat values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant.

The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

Engineering the pH-optimum of a triglyceride lipase: from predictions based on electrostatic computations to experimental results

The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein engineering endeavours. Here, we present a successful strategy for altering the pH-optimum of the triglyceride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment are correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we studied so far. This has prompted us to propose the ‘The Electrostatic Catapult Model’ as a model for product release after cleavage of the ester bond. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge mutants displayed altered titration behaviour of active site electrostatic potentials. Typically, the substitution of the residue Glu44 pushes the onset of the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the experimentally observed. Finally, it was found that the pH-dependent computed Coulombic energy displayed a strong correlation with the observed melting temperatures of native cutinase.

Orf135 from Escherichia coli Is a Nudix Hydrolase Specific for CTP, dCTP, and 5-Methyl-dCTP

Orf135 from Escherichia coli is a new member of the Nudix (nucleoside diphosphate linked to some other moiety, x) hydrolase family of enzymes with substrate specificity for CTP, dCTP, and 5-methyl-dCTP. The gene has been cloned for overexpression, and the protein has been overproduced, purified, and characterized. Orf135 is most active on 5-methyl-dCTP (k(cat)/K(m) = 301,000 M(-1) s(-1)), followed by CTP (k(cat)/K(m) = 47,000 M(-1) s(-1)) and dCTP (k(cat)/K(m) = 18,000 M(-1) s(-1)). Unlike other nucleoside triphosphate pyrophophohydrolases of the Nudix hydrolase family discovered thus far, Orf135 is highly specific for pyrimidine (deoxy)nucleoside triphosphates. Like other Nudix hydrolases, the enzyme cleaves its substrates to produce a nucleoside monophosphate and inorganic pyrophosphate, has an alkaline pH optimum, and requires a divalent metal cation for catalysis, with magnesium yielding optimal activity. Because of the nature of its substrate specificity, Orf135 may play a role in pyrimidine biosynthesis, lipid biosynthesis, and in controlling levels of 5-methyl-dCTP in the cell.

Partial purification and characterisation of a xylanase enzyme produced by a micro-organism isolated from selected indigenous fruits of Zimbabwe

Abstract Aerobic bacteria and fungi isolated from Ziziphus mauritiana, Scierocarya birrea fruits and a cattle compost were screened for production of endo-xylanase enzyme. Xylanolytic activity was found in 10 of the 88 isolates obtained. Two best endo-xylanase enzyme producers (SB-9a and TC-17d) were selected for further investigations. The two isolates were classified as belonging to the genus Bacillus. The endo-xylanase enzymes from both isolates were optimally active at pH 8 and stable over a pH range of 6.0–9.0. The optimum temperature for xylanase activity, assayed at pH 8 was 60°C. The endo-xylanase from isolate SB-9a was stable at 50°C, maintaining over 50% of its activity for 1 h at pH 8. The endo-xylanase from isolate TC-17d was less stable, maintaining about 20% of its activity for 20 mm at 50°C and pH 8. Endo-xylanase activity for isolate SB-9a was inhibited by Hg 2+ , Ag + and Mn 2+ ions while Fe 3+ , K+, Nat, Ca 2+ , and Cu 2+ ions stimulated xylanase activity. The endo-xylanase enzyme from isolate SB-9a was partially purified by ammonium sulphate precipitation, and gel filtration chromatography. It had a specific activity of 308 nkat/mg protein. This enzyme could have potential uses in biotechnological applications such as in pulp, paper and food manufacture due to its high specific activity and alkaline pH optima.

A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in crassulacean acid metabolism-induced leaves of the common ice plant.

Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric properties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the molecular cloning of PPcK from the facultative Crassulacean acid metabolism (CAM) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase-targeted differential display reverse transcriptase-polymerase chain reaction approach. M. crystallinum PPcK encodes a minimal, Ca(2+)-independent Ser/threonine protein kinase that is most closely related to calcium-dependent protein kinases, yet lacks both the calmodulin-like and auto-inhibitory domains typical of plant calcium-dependent protein kinase. In the common ice plant PPcK belongs to a small gene family containing two members. McPPcK transcript accumulation is controlled by a circadian oscillator in a light-dependent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), making it among the smallest protein kinases characterized to date. Initial biochemical analysis of the purified, recombinant McPPcK gene product documented that this protein kinase specifically phosphorylates PEPc from CAM and C(4) species at a single, N-terminal Ser (threonine) residue but fails to phosphorylate mutated forms of C(4) PEPc in which this specific site has been changed to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca(2+)-insensitive, and displayed an alkaline pH optimum. Furthermore, recombinant McPPcK was shown to reverse the sensitivity of PEPc activity to L-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynthetic carbon flux in CAM plants.

Midgut Proteinase Types and Sodium Tetraborate Effects on Midgut Proteinase Activities of Female <I>Anastrepha suspensa</I> (Diptera: Tephritidae)

Abstract Trypsin-like, chymotrypsin-like, aminopeptidase and carboxypeptidase A and B were detected in the midgut of adult female Anastrepha suspensa (Loew). Based on specific substrates and inhibitors, endoproteinases were demonstrated to be serine proteinases in which trypsin-like enzyme seemed to be the major digestive enzyme. These proteinases had alkaline pH optima, pH 7.5–9. All determined proteinase activities in the midgut were reduced to different degrees in the first few days after newly emerged flies were treated for 24 h with 0.2 and 0.5% sodium tetraborate. The 0.5% concentration caused greater reductions. These reductions in proteinase activities were not the result of direct enzyme inhibition.

Purification and characterization of a novel extracellular protease from Beauveria bassiana

An extracellular protease designated BBP (Beauveria bassiana protease) was purified from a B. bassiana isolate and subsequently characterized. It was produced in the late stages of growth in a gelatin medium, and was purified using a combination of ionexchange chromatography and preparative gel electrophoresis. Inhibition by phenylmethylsulphonyl fluoride and chymostatin indicates that BBP is a serine type with chymotrypsin activity. Substrate specificity indicates that the protease has elastase activity as well. The protease was stable at 25 °C and had an alkaline pH optimum (7.5–9.5). Another protease was purified from the same isolate that produced BBP, and was identified as Pr1 based on its isoelectric point, amino terminal sequence, substrate specificity and cuticle degrading ability. A comparison between the two showed that BBP had a lower isoelectric point (pl 7.5) than Pr1 (pl ≥ 10) and was 0.5 kDa smaller. The proteases also exhibited differences in substrate specificity with Pr1 being most active against Suc-Ala-Ala-Pro-Phe-pNA, and BBP being most active against MeOSuc-Ala-Ala-Pro-Met-pNA. The two proteases had equal cuticle-degrading activity and both were expressed in cuticle media, although the timing of their expression differed.

22] Ceramidases

Ceramidase activity was first characterized as an enzyme with an acidic pH optimum present in all rat tissues examined. This enzyme was later shown to account for a rare genetic disorder called Farber's disease where there is accumulation of ceramide in the lysosomes of spleen, cerebellum, fibroblasts, and kidney. An activity with alkaline pH optimum, however, was retained in Farber's patients, indicating that at least two ceramidases hydrolyze ceramide. Both enzymes are membrane associated; however, the acidic enzyme is localized primarily in the lysosomes and activated by Saposine D, which inhibits the alkaline enzyme (IC 50 1-5μM). The purified acid ceramidase shows little specificity with respect to the fatty acid chain length of ceramidases, but prefers sphingosine over sphinganine-containing ceramides. In addition to the acidic and alkaline activities, hydrolysis of ceramide is also found at neutral pH in rat liver, small intestine, and epithelial cells. Ceramidase (N-acylsphingosine deacylase) hydrolyzes ceramide to a free sphingoid base and fatty acid. Because ceramide can induce cell death, whereas the phosphorylated product of sphingosine, sphingosine-1-phosphate, is a potent inducer of cell proliferation and survival signals, ceramidase(s) play a key role in regulating the amounts of the bioactive sphingolipids. This chapter discusses methods for assaying ceramidases as well as factors known to participate in the regulation of these enzymes.