Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.     

Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Acaricidal activity of aqueous extracts of camomile flowers, Matricaria chamomilla, against the mite Psoroptes cuniculi

Arcaricidal properties of decoctions, infusions and macerates of dried flower heads of camomile, Matricaria chamomilla L. (Asterales: Asteraceae) were tested in vitro against the mite Psoroptes cuniculi Delafond (Parasitiformes: Psoroptidae). This mite species is responsible for otoacariasis in domestic animals. Mites were exposed to the extracts for 24, 48 or 72 h. All the extracts tested showed highly significant acaricidal activity when compared with controls. Among them, a decoction of 10% was the only formulation which gave 100% activity at all the three observations times.     

Specificity of acid proteinase A from Aspergillus niger var. macrosporus towards B-chain of performic acid oxidized bovine insulin.

1. A comparative study on the mode of action of two highly purified acid endopeptidases (EC 3.4.-) from Aspergillus niger var. macrosporus, acid proteinase A and B, on the B-chain of performic acid oxidized insulin was performed, putting emphasis on the quantitative analysis of the effects of enzyme A. Acid proteinase A behaved very specifically towards the substrate and hydrolyzed four peptide bonds exclusively: three major sites, where hydrolysis proceeded rapidly and almost completely, Asn3-Gln4, Glu13-Ala14, and Tyr26-Thr27; and a minor one, Gly20-Glu21, at which hydrolysis was much slower. 2. The effects of four protease inhibitors, pepstatin, diazoacetyl-D,L-norleucine methyl ester/Cu(II), di-isopropyl phosphorofluoridate, and 1,2-epoxy-3-(p-nitrophenozy) propane on acid proteinases A and B were studied. Acid proteinase A preparations, treated with the former two inhibitors, were used to establish that the major sites of attack were really affected by enzyme A and not by contaminating proteinase B.     

Hepatitis A virus 3C proteinase substrate specificity.

Hepatitis A virus (HAV) 3C proteinase is responsible for processing the viral precursor polyprotein into mature proteins. The substrate specificity of recombinant hepatitis A 3C proteinase was investigated using a series of synthetic peptides representing putative polyprotein junction sequences. Two peptides, corresponding to the viral polyprotein 2B/2C and 2C/3A junctions, were determined to be cleaved most efficiently by the viral 3C proteinase. The kcat/Km values determined for the hydrolysis of a further series of 2B/2C peptides, in which C-terminal and N-terminal amino acids were systematically removed, revealed that P4 through P2' amino acids were necessary for efficient substrate cleavage. The substitution of Ala for amino acids in P1 and P4 positions decreased the rate of peptide hydrolysis by 100- and 10-fold, respectively, indicating that the side chains of Gln in P1 and Leu in P4 are important determinants of substrate specificity. Rates of hydrolysis measured for other P1- and P4-substituted peptides indicate that S1 is very specific for the Gln side chain whereas S4 requires only that the amino acid in P4 be hydrophobic. A continuous fluorescence quench assay was developed, allowing the determination of kcat/Km dependence on pH. The pH rate profile suggests that catalyzed peptide hydrolysis is dependent on deprotonation of a reactive group having a pKa of 6.2 (+/- 0.2). The results of tests with several proteinase inhibitors indicate that this cysteine proteinase, like other picornaviral 3C proteinases, is not a member of the papain family.     

Protein unfolding in drug-RNase complexes

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5' bonds in RNA on the 3' side of pyrimidine to form cyclic 2', 5'-phosphates. It has several high affinity binding sites that make it possible target for many organic and inorganic molecules. Ligand binding to RNase A can alter protein secondary structure and its catalytic activity. In this review, the effects of several drugs such as AZT (anti-AIDS), cis-Pt (antitumor), aspirin (anti-inflammatory), and vitamin C (antioxidant) on the stability and conformation of RNase A in vitro are compared. The results of UV-visible, FTIR, and CD spectroscopic analysis of RNase complexes with aspirin, AZT, cis-Pt, and vitamin C at physiological conditions are discussed here. Spectroscopic results showed one major binding for each drug-RNase adduct with KAZT=5.29 (+/-1.6)x10(4) M(-1), Kaspirin=3.57 (+/-1.4)x10(4) M(-1), Kcis-Pt=5.66 (+/-1.9)x10(3) M(-1), and Kascorbate=3.50 (+/-1.5)x10(3) M(-1). Major protein unfolding occurred with reduction of alpha-helix from 29% (free protein) to 20% and increase of beta-sheet from 39% (free protein) to 45% in the aspirin-, ascorbate-, and cis-Pt-RNase complexes, while minor increase of alpha-helix was observed for AZT-RNase adduct.     

Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using [3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.     

Comparative quantitative structure toxicity relationships for flavonoids evaluated in isolated rat hepatocytes and HeLa tumor cells

Quantitative structure activity relationship (QSAR) equations were obtained to describe the cytotoxicity of 22 polyphenols using toxicity (logLD50) representing the concentration for 50% cell survival in 2 h for isolated rat hepatocytes, log P representing octanol/water partitioning, and/or E(p/2) representing redox potential. One- and two-parameter equations were derived for the quantitative structure toxicity relationships (QSTR) for polyphenol induced hepatocyte cytotoxicity: e.g. log C(hepatocyte) (microM)=-0.65(-0.08)log P+4.12(-0.15) (n=19, r(2)=0.80, s=0.33, P<1 x 10(-6)). One- and two-parameter QSAR equations were also derived to describe the inhibitory effects of 13 polyphenols on tumor cell growth when incubated with HeLa cells for 3 days: e.g. log C(tumor) (microM)=-0.34(+/-0.04)log P+2.40(+/-0.07) (n=11, r(2)=0.90, s=0.13, P<1 x 10(-5)). These findings point to lipophilicity as a major characteristic determining polyphenol cytotoxicity. The E(p/2) also played a significant role in polyphenol cytotoxicity towards both cell types: e.g. log C(hepatocyte) (microM)=-0.60(+/-0.06)log P+2.01(+/-0.43)E(p/2) (V)+3.86(+/-0.12) (n=9, r(2)=0.96, s=0.15, P<0.005). The involvement of log P and E(p/2) could be explained if polyphenol cytotoxicity involved the formation of radicals, which interacted with the mitochondrial inner membrane resulting in a disruption of the membrane potential.     

Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Enzyme activities of cell-free extracts from mutant strains of lactic streptococci subjected to sublethal heating or freeze-thawing

Two mutant lactose-negative (Lac-), proteinase-negative (Prt-) strains of lactic streptococci, Streptococcus lactis 25Sp and S. cremoris KHA2, and their parents, S. lactis C2 and S. cremoris KH Lac+ Prt+, were grown in a suitable medium with the pH maintained at 6.5 by addition of NH4OH. Cells were harvested by centrifugation, resuspended, and then heated sublethally at 54 or 69 degrees C for 15 sec. Cells also were frozen and stored for 1 week at -20 or -100 degrees C. Cell-free extracts of cells heated at 54 degrees C had more proteinase and aminopeptidase activities than did a similar extract of cells heated at 69 degrees C. The greatest enzyme activities occurred in the cell-free extracts prepared from cells frozen and stored at -100 degrees C. Specific activities of proteinase and dipeptidase generally decreased in extracts of freeze-shocked cells compared to those in extracts of untreated cells. Enzyme activity of extracts also decreased in the presence of 5% NaCl at pH 5.0. Cell-free extracts at pH values of 5 to 8 were heated at 69 degrees C for 1.5, or 10 min. Heating them for 10 min caused a loss of dipeptidase activity which was most pronounced at pH 5.0 and least pronounced at pH 7.0.     

Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae)

Digestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.     

The role of proteolytic enzymes in protein degradation during senescence of rice leaves

The role of proteolytic enzymes in protein degradation of detached and intact leaves of rice seedling ( Oryza sativa L. cv. Taiching Native 1) during senescence and of mature leaves during reproductive development was investigated. The amount of soluble protein decreased by about 50% in 2, 4, and 15 days for detached, intact and mature leaves, respectively. Three proteolytic enzyme activities were monitored with pH optima of 4.5 for hemoglobin-digesting proteinase, 5.5 for carboxypeptidase and 8.0 for aminopeptidase. No azocoll-digesting proteinase activity could be detected in rice leaves. Dialysis did not alter the activities of any of the three proteolytic enzymes. Acid proteinase activity and aminopeptidase activity were highly unstable during storage of the enzyme extracts at 4°C. Proteolysis was stimulated by inclusion of meroaptoethanal either in the extraction medium or the assay medium.
Acid proteinase, carboxypeptidase and aminopeptidase were all present in detached, intact and mature leaves throughout senescence. There seems to be a direct correlation between protein degradation and increases of acid proteinase and carboxypeptidase activity in seedling leaves (detached and intact) during senescence. In senescing (detached and intact) leaves of seedlings the acid proteinase activity developed first, while that of carboxypeptidase developed later. Acid proteinase and carboxypeptidase may play major roles in protein degradation of leaves from seedlings during senscence. During reproductive development, protein degradation was associated with decreases in the activities of acid proteinase, carboxypeptidase and aminopeptidase in mature leaves suggesting that the enzymes were less important for protein degradation in this system. Hence, the role of protelytic enzymes in protein degradation during senescence of rice leaves appears to depend largely on the leaf system used.     

Bioactivity of the metabolites from endophytic actinomycete Fq24 against Tetranychus cinnabarinus

An endophytic actinomycete strain Fq24 was isolated from healthy tomato plants. The acaricidal substances in the metabolites from Fq24 were collected and identified by the methods of extraction, column chromatography, and gas chromatography-mass spectrometry (GC-MS), and their bioactivities against Tetranychus cinnabarinus were measured with slide-dip and leaf-residue methods. Among the extracts, petroleum ether extract had high bioactivity in contact toxicity and oviposition deterrent against T. cinnabarinus. Its lethal concentration of 50% (LC50) was 52.57 mg x L(-1), and its oviposition deterrent concentration of 50% (ODC50) was 43.18 mg x L(-1). The identification with GC-MS showed that the main chemical component of fraction S11 was methyl hexadecanoate, whose molecular formula was C17H34O2, being one of the substances with acaricidal activity in the metabolites from Fq24. The 24 h corrected mortality rate of female mite at 5 mg x mL(-1) of methyl hexadecanoate was 78.3%, and the oviposition deterrent rate was 81.6%.     

Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1). Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.     

Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells.

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).     

The interaction between severe acute respiratory syndrome coronavirus 3C-like proteinase and a dimeric inhibitor by capillary electrophoresis

3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been demonstrated to be a key target for drug design against SARS. The interaction between SARS coronavirus 3C-like (3CL) proteinase and an octapeptide interface inhibitor was studied by affinity capillary electrophoresis (ACE). The binding constants were estimated by the change of migration time of the analytes in the buffer solution containing different concentrations of SARS 3CL proteinase. The results showed that SARS 3CL proteinase was able to complex with the octapeptide competitively, with binding constants of 2.44 x 10(4) M(-1) at 20 degrees C and 2.11 x 10(4)M(-1) at 37 degrees C. In addition, the thermodynamic parameters deduced reveal that hydrophobic interaction might play major roles, along with electrostatic force, in the binding process. The ACE method used here could be developed to be an effective and simple way of applying large-scale drug screening and evaluation.     

Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors

Zinc binding to the two Cys(4) sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co(2+) as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys(4) structural zinc-binding sites found in the DBD of human estrogen receptor alpha (hERalpha-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co(2+) to the two proteins was determined relative to the binding affinity of Co(2+) to the zinc finger consensus peptide, CP-1. Using the known dissociation constant of Co(2+) from CP-1, the dissociation constants of cobalt from hERalpha-DBD were calculated: K(d1)(Co) = 2.2 (+/- 1.0) x 10(-7) M and K(d2)(Co) = 6.1 (+/- 1.5) x 10(-7) M. Similarly, the dissociation constants of Co(2+) from GR-DBD were calculated: K(d1)(Co) = 4.1 (+/- 0.6) x 10(-7) M and K(d2)(Co) = 1.7 (+/- 0.3) x 10(-7) M. Metal-binding studies conducted in which Zn(2+) displaces Co(2+) from the metal-binding sites of hERalpha-DBD and GR-DBD indicate that Zn(2+) binds to each of the Cys(4) metal-binding sites approximately 3 orders of magnitude more tightly than Co(2+) does: the stoichiometric dissociation constants are K(d1)(Zn) = 1 (+/- 1) x 10(-10) M and K(d2)(Zn) = 5 (+/- 1) x 10(-10) M for hERalpha-DBD and K(d1)(Zn) = 2 (+/- 1) x 10(-10) M and K(d2)(Zn) = 3 (+/- 1) x 10(-10) M for GR-DBD. These affinities are comparable to those observed for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative [Low, L. Y., Hernández, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERalpha-DBD and GR-DBD do not interact.     

The accessibility of iron at the active site of recombinant human phenylalanine hydroxylase to water as studied by 1H NMR paramagnetic relaxation. Effect of L-Phe and comparison with the rat enzyme

The high-spin (S = 5/2) Fe(III) ion at the active site of recombinant human phenylalanine hydroxylase (PAH) has a paramagnetic effect on the longitudinal relaxation rate of water protons. This effect is proportional to the concentration of enzyme, with a paramagnetic molar-relaxivity value at 400 MHz and 25 degrees C of 1. 3 (+/- 0.03) x 10(3) s-1 M-1. The value of the Arrhenius activation energy (Ea) for the relaxation rate was -14.4 +/- 1.1 kJ/mol for the resting enzyme, indicating a fast exchange of water protons in the paramagnetic environment. The frequency dependence of the relaxation rate also supported this hypothesis. Thus, the recombinant human PAH appears to have a more solvent-accessible catalytic iron than the rat enzyme, in which the water coordinated to the metal is slowly exchanging with the solvent. These findings may be related to the level of basal activity before activation for these enzymes, which is higher for human than for rat PAH. In the presence of saturating (5 mM) concentrations of the substrate L-Phe, the paramagnetic molar relaxivity for human PAH decreased to 0.72 (+/- 0.05) x 10(3) s-1 M-1 with no significant change in the Ea. Effective correlation times (tauC) of 1.8 (+/- 0.3) x 10(-10) and 1.25 (+/- 0.2) x 10(-10) s-1 were calculated for the enzyme and the enzyme-substrate complex, respectively, and most likely represent the electron spin relaxation rate (tauS) for Fe(III) in each case. Together with the paramagnetic molar-relaxivity values, the tauC values were used to estimate Fe(III)-water distances. It seems that at least one of the three water molecules coordinated to the iron in the resting rat and human enzymes is displaced from coordination on the binding of L-Phe at the active site.     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.