Digestive proteases in the midgut gland of the atlantic blue crab,Callinectes sapidus

• 1.1. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.
• 2.2. Trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase activities were found and quantified.
• 3.3. Activity against Succinyl-(Ala)₃-nitroanalide was also found. This as yet unidentified enzyme has a mol. wt of about 26,000 and has elastolytic activity.

     

Properties of extracellular plasmin-like proteases of <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> micromycete

The properties of two extracellular proteases of Aspergillus ochraceus VKM F-4104D micromycete with plasmin-like activity have been studied. It has been shown that the enzymes differ in pI (5.05 and 6.83) and have similar molecular weights (about 32 and 35 kDa), pH optima (pH 9.0–10.00 at 45°C), and specificities of action on a limited set of chromogenic peptide substrates of trypsin-like proteases. According to inhibitory analysis, both enzymes belong to the serine proteases. Their properties appeared to be similar to those of the protease, protein C activator, which is the main proteolytic enzyme of A. ochraceus VKM F-4104D. Most likely, proteases of this micromyсetes are isoenzymes.     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60–85%) and the γ-subunit of nerve growth factor (by 55–93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the γ-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), IV), and (VI)], papain [(I), (II), IV), and (VI)], subtilisin [(V) and (VI)], α-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 × 10^?2 M, while, at the concentration of 1 × 10^?3 M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

Characterization and partial purification of the digestive proteases of the black field cricket,Teleogryllus commodus (Walker): Elastase is a major component

The major proteases of the black field cricket, Telleogryllus commodus, digestive system have been identified, partially purified and characterized. Classification of proteases into different classes of endo- and exopeptidases was made on the ability to hydrolyse specific synthetic substrates, pH optima and their interaction with a range of specific chemical and proteinaceous inhibitors. The major activities detected were trypsin, elastase, an uncharacterized proteinase (proteinase Tc), leucine aminopeptidase and carboxypeptidases A and B. Chymotrypsin activity was very low and neither cysteine endopeptidase nor metalloendopepitidase activities were found. Elastase is a newly discovered protease activity for insects.Trypsin, elastase and proteinase Tc have molecular weights of 24,300, 19,500 and 23,600, respectively; show alkaline pH optima and chemical inhibition indicative of serine endopeptidases; and interact most strongly with their characteristic class of proteinaceous inhibitors. Elastase and proteinase Tc are inhibited by a very similar spectrum of specific inhibitors, but the latter lacks activity against all specific synthetic substrates tested. Leucine aminopeptidase and carboxypeptidase A have molecular weights of 94,000 and 39,700, respectively, and show optimum activity at pH 8 and pH 9, respectively.The equilibrium dissociation constants for trypsin, elastase and proteinase Tc with 25 serine proteinase inhibitors were measured. Values spanning a 1000-fold range were obtained in each case.     

Enzyme antigens associated with pigeon breeder's disease. II. Isolation and characterization of acidic hydrolases

The hydrolytic enzymes in pigeon dropping extracts (PDE) were separated into basic and acidic components by DEAE-cellulose chromatography. Six distinct hydrolytic activities were isolated from the acidic group of enzymes. Elastase, trypsin, and two forms of collagenase were the proteases identified. An esterase and a phospholipase were also detected. The sera of symptomatic pigeon breeders, analyzed by crossed immunoelectrofocusing techniques, were shown to contain antibodies to the enzymes trypsin, collagenase, and esterase in the acidic fractions of PDE by staining the immune precipitates with specific chromogenic substrates. These enzymes were purified by preparative isoelectric focusing, affinity chromatography, and gel filtration. The purified elastase hydrolyzed elastin and consisted of a single polypeptide chain (M _r =23,100). Trypsin hydrolyzed a synthetic arginine substrate, but not elastin or collagen. Its size (M _r =27,500) and subunit structure were similar to those reported here for elastase. Both enzymes were inhibited by -1-antitrypsin,N-p-tosyl-L-lysine chloromethylketone, and phenylmethylsulfonyl fluoride. Two distinct collagenases were found; both cleaved bovine collagen. The high-molecular-weight (HMW) collagenase was a glycoprotein (M _r =117,500) and consisted of three polypeptide chains (M _r =37,100); a low-molecular-weight (LMW) collagenase (M _r =12,500) was also isolated. The HMW collagenase was inhibited byEDTA,p-chloromercuribenzoic acid, and phenylmethylsulfonyl fluoride, but not by -1-antitrypsin. The source of these enzymes as well as their relationship to the basic hydrolytic enzymes of PDE are discussed.     

Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties

Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca²⁺ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The K_m values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P₁ position.     

Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family

A chemoenzymatic synthesis was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are o-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 × 10^?10 M for (I) and 1.2 × 10^?11 M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Comparison of bovine β-casein hydrolysis by PI and PIII-type proteinases from Lactobacillus lactis subsp. cremoris

Summary The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine -casein was compared. The proteinase from the H2 strain was characterised as a P_I-type proteinase since it did not hydrolyse _s1-casein and the initial trifluoroacetic acid-soluble products of -casein hydrolysis were identical to those previously identified as hydrolysis products of P_I-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr₁₉₃-Gln₁₉₄ and Gln₁₈₂-Arg₁₈₃ were the first to be hydrolysed. Cleavage of the bonds Gln₁₇₅-Lys₁₇₆, Ser₁₆₈-Lys₁₆₉, Ser₁₆₆-Gln₁₆₇ and Leu₁₆₃-Ser₁₆₄ was also very rapid. Four of the five bonds in -casein most susceptible to hydrolysis by the P_III-type proteinase from strain SK112 were different from those cleaved by the P_I-type proteinase, initial hydrolysis being at the sites Tyr₁₉₃-Gln₁₉₄, Leu₁₉₂-Tyr₁₉₃, Asp₄₃-Glu₄₄, Gln₄₆-Asp₄₇ and Phe₅₂-Ala₅₃. Early hydrolysis at the three sites in the N-terminal region of -casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of P_I-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly. Offprint requests to: G. G. Pritchard     

Purification and characterization of proteases from the polychaete annelid Sabellaria alveolata (L.)

Eleven proteases have been purified to electrophoretic homogeneity from crude digestive fluid of polychaete annelids, Sabellaria alveolata. Purification steps were Sephadex G-100 gel filtration, benzamidine-cellulose and SBTI-Sepharose (SBTI = soybean trypsin inhibitor) affinity chromatography, CM-Sepharose and DEAE-Sepharose ion-exchange chromatography. Nine proteases have been purified in sufficient quantities for characterization. All are active at basic pH and are probably serine proteases, since they are inhibited by phenylmethylsulfonyl fluoride, specific chloromethyl ketone amino acids derivatives, but not by EDTA and p-chloromercuribenzoate. They do not hydrolyse exopeptidase substrates. From their properties, they can be divided into five classes. 1. A trypsin-like protease, which hydrolyses only trypsin substrates and is inhibited by N-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl), leupeptin and antipain. It differs from bovine trypsin by its very acidic isoelectric point (below 3.3) and its higher Mr (35 000). 2. A chymotrypsin-like protease which hydrolyses only chymotrypsin substrates and is inhibited by TosPheCH2Cl, Z-PheCH2Cl, chymostatin but only slightly by leupeptin and antipain. Its isoelectric point is below 3.3 and its Mr 31 000. 3. Two minor chymotrypsin-like proteases with slightly broader specificity, since they hydrolyse trypsin substrates significantly and are much more inhibited by leupeptin. They have acidic isoelectric points (3.3 and 3.5) and slightly lower Mr (27 000). 4. Four proteases hydrolyse trypsin and chymotrypsin substrates equally well. Their chymotryptic character is, however, predominant since they are inhibited by TosPheCH2Cl and Z-PheCH2Cl but not TosLysCH2Cl. They have similar Mr (27 000) but isoelectric points ranging from 4.0 to above 9.1. 5. The last one is very similar but has lower esterolytic activities. These proteases of broad specificity do not resemble any known serine protease since they differ from subtilisins by their sensitivity to TosPheCH2Cl.     

A computational approach to design energetic ionic liquids

The present work deals with the theoretical estimation of ion-pair binding energies and the energetic properties of four ion pairs formed by combining the 1-butyl-2,4-dinitro-3-methyl imidazolium ion with nitrate (I), perchlorate (II), dinitramide (III), or 3,5-dinitro-1,2,4-triazolate (IV) anions. The counterpoise-corrected ion-pair binding energies were calculated for each ion pair at the B3LYP/6-311+G(d,p) level of theory. Results show that the cation–anion interaction is strongest for ion pair I and weakest for IV, indicating that the nitrate (I) has a greater tendency to exist as a stable ionic salt whereas the 3,5-dinitro-1,2,4-triazolate (IV) may exist as an ionic liquid. Natural bond orbital (NBO) analysis and electrostatic potential (ESP) mapping revealed that charge transfer occurs in all of the ion pairs, but is greatest (0.25e) for ion pair I and smallest (0.03e) for IV, resulting in ion pair I being the least polarized. A nucleus-independent chemical shift (NICS) study revealed that the aromaticity of the 1-butyl-2,4-dinitro-3-methyl imidazolium ion significantly increases in ion pair IV, indicating that this has the greatest charge delocalization among all of the four ion pairs considered. Studies of thermodynamic and detonation properties showed that ion pair II is the most energetic ion pair in terms of its detonation velocity (D = 7.5 km s^?1) and detonation pressure (P = 23.1 GPa). It is also envisaged that ion pair IV would exist as an energetic azolium azolate type ionic liquid that could be conveniently used as a secondary explosive characterized by detonation parameters D and P of 6.9 km s^?1 and 19.3 GPa, respectively. These values are comparable to those of conventional explosives such as TNT.     

Protein breakdown and protease properties of germinating maize endosperm

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G₃ glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P₁₁, P₂₁ and P₂₂) have been isolated by affinity chromatography and gel filtration, and partially characterized. P₁₁ (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P₂₁ (MW 36 000) and P₂₂ (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro. Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.     

Synthesis, spectral (UV-Vis, IR, ESI-MS), magnetic and structural characterizations, and the antimicrobial effect of potassium isothiocyanato-(N-salicylidene-amino-acidato)cuprates

A series of potassium isothiocyanato-(N-salicylidene-amino acidato)cuprates with the general formulas of K₂[Cu₂(sal-aa)₂(μ-NCS)₂]·nH₂O, where n = 0 or 4 and (sal-aa) stands for the dianion of N-salicylideneamino acid derived from glycine (I), dl-α-alanine (II), dl-valine (III), dl-phenylalanine (IV), and {K[Cu(sal-β-ala)(μ-NCS)]}_n for β-alanine (V), respectively, was synthesized and fully characterized by elemental analysis, UV-Vis and IR spectroscopy, ESI-MS spectrometry, magnetic measurements, and X-ray structural analysis (II and IV). It has been found that the copper(II) atom adopts a distorted square-pyramidal surrounding in the dimeric complexes I-IV, while the geometry in the polymeric complex V can be described as distorted square-bipyramidal. The analysis of magnetic properties revealed weak antiferromagnetic exchanges in the dinuclear species I-IV and an alternating ferro/antiferromagnetic exchange in the case of 1D-polymeric compound V. Moreover, the complexes were tested for their antibacterial activity against the G+ bacteria Staphylococcus aureus, G− bacteria Escherichia coli, filamentous fungi Microsporum gypseum, and yeast Candida albicans. The best results were achieved with G+ bacteria S. aureus with MIC values in the range of 0.22-0.57 mmol L⁻¹. It may be concluded that both the antimicrobial and antifungal activity decreased within the tested group of cuprates derived from α-amino acids with the increasing lipophility of the complexes, i.e.I → IV.     

Multiple Forms of α-Glucosidase from Pig Duodenum

Multiple forms of neutral α-glucosidase (pH optima, 6.0~6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glueosidases I_a and II_a were 145,000~150,000, pH 3.5~3.7 and 55°C, respectively, and both enzymes were stable up to 55°C on treatment at pH 6.0 for 15 min; whereas those of the other two α-glucosidases, I_b and II_b, were 80,000, pH 4.0~4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mm, 0.37 mm and 1.67mg/ml, while those of enzyme II_b were 3.33 mm, 2.61 mm and 11.8 mg/ml, respectively. All enzyme hydrolyzed α-1,4-, α-1,3- and α-1,2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.     

The regional and subcellular distribution of calcium activated neutral proteinase (CANP) in the bovine central nervous system

Calcium-activated neutral proteinase (CANP) activity was determined in subcellular fractions and in different regions of bovine brain. The CANP specific activity in spinal cord and corpus callosum, areas rich in myelin, were almost six-fold greater than cerebral cortex and cerebellum. Treatment of whole homogenate and myelin with 0.1% Triton X-100 increased the CANP activity by tenfold. Subcellular fractions were prepared from bovine brain gray and white matter. Most of the CANP activity (70%) was in the primary particulate fractions P₁ (nuclear), P₂ (mitochondrial) and P₃ (microsomal). On subfractionation of each particulate fraction, the majority of the activity (greater than 50%) was recovered in the myelin-enriched fractions (P₁A, P₂A, P₃A) which separate at the interphase of 0.32 M- and 0l85 M-sucrose. The distribution of activity was P₂A>P₁A>P₃A. Further purification of myelin (of P₂A) increased the specific activity over homogenate by more than three-fold. The same myelin fractions contained the highest proportion (60%) and specific activity (five-fold increase) of CNPase. The enzyme activity in different regions of brain and in subcellular fractions was increased by 20–39% after the inhibitor was removed. Electron microscopic study confirmed that the myelin fractions were highly purified. The cytosolic fraction contained 20–30% of the total homogenate CANP activity. Other fractions contained low enzyme activity. CANP was identified in the purified myelin fraction by electroimmublot-technique. It is concluded that the bulk of CANP in CNS is tightly bound to the membrane, may be masked or hidden and is intimately associated with the myelin sheath.Abbreviations Used CANP calcium-activated neutral proteinase - CNPase adenosine-2, 3-cyclic nucleotide 3-phosphohydrolase     

The midgut trypsins of shrimp (Penaeus monodon). High efficiency toward native protein substrates including collagens

Two shrimp trypsins have been purified from the midguts of Penaeid shrimps by various chromatographies and HPLC. The molecular masses of them are 27 and 29 kDa, respectively. They show the typical specificity of trypsin for substrates and inhibitors, and their N-terminal amino-acid sequences are homologous to those of other trypsins. The shrimp enzymes are very acidic (pI less than or equal to 2.4), and show distinctively low Km for the synthetic amide substrates. They also hydrolyse various native proteins more efficiently than bovine trypsin in vitro. However, the anionic shrimp trypsins do not have special preference for basic protein substrates over the acidic one. Collagenolytic activity of the midgut extract was mainly due to serine proteases. The collagenolytic activity of the purified shrimp trypsin was confirmed by assays with either soluble or insoluble native type I collagens. In comparison with the other trypsins from the Crustacean decapods, the shrimp enzymes have four pairs of disulfide bonds, intermediary between the crayfish trypsin (three pairs) and the crab trypsin (five pairs), and are immunochemically different from them.     

Fractionation of the chymotryptic precipitate of Bombyx mori silk fibroin

1. A solution of Bombyx mori silk fibroin was digested with chymotrypsin. Amino acid analyses of the chymotryptic precipitate showed in addition to the main constituents Gly, Ala, Ser and Tyr, very small amounts of Lys, His, Arg, Asp, Thr, Glu, Pro, Cys, Val, Met, Ile, Leu, Phe and Trp. 2. A stable solution of the chymotryptic precipitate in 6m-urea was obtained by dialysing a solution in 50% (w/v) lithium thiocyanate against 6m-urea. 3. The dinitrophenylated chymotryptic precipitate in 6m-urea was fractionated by gel filtration and by ion-exchange chromatography. On Dowex 1 (X2), a main fraction I_d and three further fractions with different amino acid compositions and molecular weights were obtained. 4. Specific rearrangement and fission of the bonds involving the serine nitrogen atoms of fraction I_d and fractionation of the resulting mixture by gel filtration yielded five fractions. Two of these fractions had the compositions DNP-Ser-(Gly₆,Ala₄,Ser) and DNP-Ser-(Gly₄,Ala₂ or Ala₃,Ser) and are presumably double repeating units according to the proposed formula of Lucas, Shaw & Smith (1957), namely [Ser-Gly-(Ala-Gly)_n]₂, for n values of 2 and 1 respectively.     

Cold-Adapted Digestive Aspartic Protease of the Clawed Lobsters Homarus americanus and Homarus gammarus: Biochemical Characterization

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K _M value at 4°C, 10°C, and 25°C, and had 20-fold higher k _cat /K _M values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.     

Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members

Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1–6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k_cat/K_m when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.