An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5′-phosphate dsDNA (5′P DNA) and degrades it without sequence specificity, but does not recognize 5′OH DNA. As is the case for most DNases, Mg²⁺ was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm.     

Hydrolytic enzymes of Psoroptes cuniculi (Delafond)

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC50 8.02 x 10(-11) M (+/- 0.30 x 10(-11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K(m) and Vmax values were 7.2 x 10(-5) (+/- 0.7 x 10(-5) M-1 and 1.13 x 10(-3) (+/- 0.05 x 10(-3) OD unit-1 min-1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21-24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.     

Participation of electrogenic Na+-K+-ATPase in the membrane potential of earthworm body wall muscles

The effect of Na+-K+-ATPase inhibitor ouabain on the resting membrane potential (Vm) was studied by glass microelectrodes in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris and compared with frog sartorius muscle. In earthworm muscle, Vm was -49 mV (inside negative) in a reference external solution with 4 mmol/l K+. The electrogenic participation of Na+-K+-ATPase was absent in solutions with very low concentrations of 0.01 mmol/l K+, higher in 4 and 8 mmol/l K+ (4-5 mV) and maximal (13 mV) in solutions containing 12 mmol/l K+ where Vm was -46 mV in the absence and -33 mV in the presence of 1 x 10(4) M ouabain. The electrogenic participation of Na+-K+-ATPase was much smaller in m. sartorius of the frog Rana temporaria bathed in 8 and 12 mmol/l K+. The results indicate that the Na+-K+-ATPase is an important electrogenic factor in earthworm longitudinal muscle fibres and that its contribution to Vm depends directly on the concentration of K+ in the bathing solution.     

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.     

Relationship of exo-beta-D-galactofuranosidase kinetic parameters to the number of phosphodiesters in Penicillium fellutanum peptidophosphogalactomannan: enzyme purification and kinetics of glycopeptide and galactofuran chain hydrolysis

Extracellular Penicillium fellutanum exo-beta-D-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP(2)GM(ii) and pP(25)GM(ii) (containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-beta-D-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP(2)GM(ii) or pP(25)GM(ii). The k(cat)/K(m) value for pP(25)GM(ii) is 1.7 x 10(3) M(-1) s(-1), that for 1-O-methyl-beta-D-galactofuranoside is 1.1 x 10(4) M(-1) s(-1), that for pP(2)GM(ii) is 1.7 x 10 (4) M(-1) s(-1), and those for 5-O-beta-D-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 x 10(5) and 4.1 x 10(5) M(-1) s(-1), respectively. Variability in the k(cat)/K(m) values is due primarily to differences in K(m) values; the k(-1)/k(1) ratio likely provides the most influence on K(m). k(cat) increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP(x)GM(ii) added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml(-1)h(-1). No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP(x)GM(ii) and of related polymers is proportional to the k(cat)/K(m) value of each polymer. The kinetic data show that the k(cat)/K(m) value increases as the number of phosphodiesters of pP(x)GM(ii) decreases, also resulting in an increase in the activity of exo-beta-D-galactofuranosidase.     

Contribution of enzyme-phosphoribosyl contacts to catalysis by orotidine 5'-phosphate decarboxylase

The crystal structure of the complex formed between recombinant yeast orotidine 5'-phosphate decarboxylase and the competitive inhibitor 6-hydroxyuridine 5'-phosphate reveals the presence of four hydrogen bonds between active site residues Tyr-217 and Arg-235 and the phosphoryl group of this inhibitor. When Tyr-217 and Arg-235 are individually mutated to alanine, values of k(cat)/K(m) are reduced by factors of 3000- and 7300-fold, respectively. In the Y217A/R235A double mutant, activity is reduced more than 10(7)-fold. Experiments with highly enriched [(14)C]orotic acid show that when ribose 5'-phosphate is deleted from substrate orotidine 5'-phosphate, k(cat)/K(m) is reduced by more than 12 orders of magnitude, from 6.3 x 10(7) M(-1) s(-1) for OMP to less than 2.5 x 10(-5) M(-1) s(-1) for orotic acid. Activity toward orotate is not "rescued" by 1 M inorganic phosphate. The K(i) value of ribose 5'-phosphate, representing the part of the natural substrate that is absent in orotic acid, is 8.1 x 10(-5) M. Thus, the effective concentration of the 5'-phosphoribosyl group, in stabilizing the transition state for enzymatic decarboxylation of OMP, is estimated to be >2 x 10(8) M, representing one of the largest connectivity effects that has been reported for an enzyme reaction.     

The spectral and paramagnetic properties of oxyhemoglobin solutions UV-irradiated in the presence of ascorbic acid]

Absorption spectra and ESR of aqueous and aqueous/glyceric solutions of oxyhemoglobin exposed to UV radiation (250-400 nm) at 293 and 77 K in the presence of ascorbic acid have been analyzed. Vitamin C (5 x 10(-5) M) has been shown to exert a photoprotective effect with regard to oxyhemoglobin (2 x 10(-6) M) UV-irradiated with a dose of 0.86 x 10(5) J/m2 at 293 K. The photoprotective effect of ascorbic acid is also displayed after UV irradiation of frozen (77 K) aqueous/glyceric oxyhemoglobin solutions (2.53 x 10(-5) M). It is concluded that ascorbic acid can be a scavenger with respect to active UV-induced particles in protein systems, including O2-. and OH. Proposed is a mode of processes leading to UV inactivation of hemoprotein molecules.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

Isolation and characterization of two digestive trypsin-like proteinases from larvae of the stalk corn borer, Sesamia nonagrioides

Two digestive trypsin-like proteinases from Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae) larvae were purified by benzamidine-Sepharose affinity chromatography. The purified enzymes showed molecular size of 27 (trypsin-I) and 24 KDa (trypsin-II). Amino acid analysis and N-terminal sequencing confirmed their relationship with other trypsins from lepidopteran larvae. However, trypsin-I presented one lysine at position 11, being the first report of this amino acid in the sequence of a lepidopteran digestive trypsin. Trypsin-I had an isoelectric point of 6.0, and a Km of 2.2 x 10(-4) M and 3.9 x 10(-5) M for BApNa and BAEE, respectively. Trypsin-II presented an isoelectric point of 8.7, and Km values of 1.7 x 10(-4) M (BApNa) and 3.8 x 10(-5) M (BAEE). Both enzymes were differentially inhibited by some proteinase inhibitors. In particular, trypsin-I was inhibited by E-64 (ID50 = 6 microM) but not by lima bean trypsin inhibitor (LBI), whereas trypsin-II was inhibited by LBI (ID50 = 1 microM) and poorly by E-64 (ID50 = 85 microM). Changes in the susceptibility of the trypsin-like activity of midgut extracts from different larval instars to these inhibitors suggest that the relative proportion of these two enzymes varied through larval development, being predominant in early instars trypsin-I and in late instars trypsin-II.     

Dimerization of ferulic and caffeic acids by purified peroxidase isolated from Bupleurum salicifolium callus culture

A novel peroxidase that catalyses the transformation of caffeic acid and ferulic acid via oxidative coupling was purified from callus cultures of Bupleurum salicifolium petioles. The enzyme, which was purified over 2,900-fold, is a glycoprotein with a molecular weight of 38,000, determined by SDS/PAGE and gel filtration. The K(m) values obtained were 2.4x10(-4) M for caffeic and 2.6x10(-4) M for ferulic acid, while the K(m) values for H2O2 with caffeic acid was 4x10(-5) M and for H2O2 with ferulic acid was 4.8x10(-4) M. The purified peroxidase exhibits lower activity with typical peroxidase substrates (guaiacol and pyrogallol) than it does with caffeic and ferulic acids, but does not exhibit any activity with other phenylpropanoids tested (cinnamic acid, coumaric acid, and 3,4-dimethoxycinnamic acid).     

Structural analysis of DNA interactions with biogenic polyamines and cobalt(III)hexamine studied by Fourier transform infrared and capillary electrophoresis

Biogenic polyamines, such as putrescine, spermidine, and spermine are small organic polycations involved in numerous diverse biological processes. These compounds play an important role in nucleic acid function due to their binding to DNA and RNA. It has been shown that biogenic polyamines cause DNA condensation and aggregation similar to that of inorganic cobalt(III)hexamine cation, which has the ability to induce DNA conformational changes. However, the nature of the polyamine.DNA binding at the molecular level is not clearly established and is the subject of much controversy. In the present study the effects of spermine, spermidine, putrescine, and cobalt(III)hexamine on the solution structure of calf-thymus DNA were investigated using affinity capillary electrophoresis, Fourier transform infrared, and circular dichroism spectroscopic methods. At low polycation concentrations, putrescine binds preferentially through the minor and major grooves of double strand DNA, whereas spermine, spermidine, and cobalt(III)hexamine bind to the major groove. At high polycation concentrations, putrescine interaction with the bases is weak, whereas strong base binding occurred for spermidine in the major and minor grooves of DNA duplex. However, major groove binding is preferred by spermine and cobalt(III)hexamine cations. Electrostatic attractions between polycation and the backbone phosphate group were also observed. No major alterations of B-DNA were observed for biogenic polyamines, whereas cobalt(III)hexamine induced a partial B --> A transition. DNA condensation was also observed for cobalt(III)hexamine cation, whereas organic polyamines induced duplex stabilization. The binding constants calculated for biogenic polyamines are K(Spm) = 2.3 x 10(5) M(-1), K(Spd) = 1.4 x 10(5) M(-1), and K(Put) = 1.02 x 10(5) M(-1). Two binding constants have been found for cobalt(III)hexamine with K(1) = 1.8 x 10(5) M(-1) and K(2) = 9.2 x 10(4) M(-1). The Hill coefficients indicate a positive cooperativity binding for biogenic polyamines and a negative cooperativity for cobalt(III)hexamine.     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.     

Vibrio harveyi NADPH-FMN oxidoreductase arg203 as a critical residue for NADPH recognition and binding

Luminous bacteria contain three types of NAD(P)H-FMN oxidoreductases (flavin reductases) with different pyridine nucleotide specificities. Among them, the NADPH-specific flavin reductase from Vibrio harveyi exhibits a uniquely high preference for NADPH. In comparing the substrate specificity, crystal structure, and primary sequence of this flavin reductase with other structurally related proteins, we hypothesize that the conserved Arg203 residue of this reductase is critical to the specific recognition of NADPH. The mutation of this residue to an alanine resulted in only small changes in the binding and reduction potential of the FMN cofactor, the K(m) for the FMN substrate, and the k(cat). In contrast, the K(m) for NADPH was increased 36-fold by such a mutation. The characteristic perturbation of the FMN cofactor absorption spectrum upon NADP(+) binding by the wild-type reductase was abolished by the same mutation. While the k(cat)/K(m,NADPH) was reduced from 1990 x 10(5) to 46 x 10(5) M(-1) min(-1) by the mutation, the mutated variant showed a k(cat)/K(m,NADH) of 4 x 10(5) M(-1) min(-1), closely resembling that of the wild-type reductase. The deuterium isotope effects (D)V and (D)(V/K) for (4R)-[4-(2)H]-NADPH were 1.7 and 1.4, respectively, for the wild-type reductase but were increased to 3.8 and 4.0, respectively, for the mutated variant. Such a finding indicates that the rates of NADPH and NADP(+) dissociation in relation to the isotope-sensitive redox steps were both increased as a result of the mutation. These results all provide support to the critical role of the Arg203 in the specific recognition and binding of NADPH.     

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.     

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.     

A kinetic analysis of the tissue plasminogen activator and DSPAalpha1 cofactor activities of untreated and TAFIa-treated soluble fibrin degradation products of varying size

The kinetics of tissue plasminogen activator (t-PA) and DSPAalpha1-catalyzed plasminogen activation using untreated and TAFIa-treated fibrin degradation products (FDPs), ranging in weight average molecular weight (M(w)) from 0.48 x 10(6) to 4.94 x 10(6) g/mol, were modeled according to the steady-state template model. The FDPs served as effective cofactors for both activators. The intrinsic catalytic efficiencies of both t-PA (17.4 x 10(5) m(-1) s(-1)) and DSPAalpha1 (6.0 x 10(5) m(-1) s(-1)) were independent of FDP M(w). The intrinsic catalytic efficiency of t-PA was 12-fold higher than that measured under identical conditions with intact fibrin as the cofactor. At sub-saturating levels of cofactor and substrate, rates were strongly dependent on FDP M(w) with DSPAalpha1 but not t-PA. Loss of activity with decreasing FDP M(w) correlated with loss of finger-dependent binding of the activators to the FDPs. TAFIa treatment of the FDPs resulted in 90- and 215-fold decreases in the catalytic efficiencies of t-PA (0.20 x 10(5) m(-)(1) s(-1)) and DSPAalpha1 (0.028 x 10(5) m(-1) s(-1)), yielding cofactors that were still 30- and 50-fold better than fibrinogen with t-PA and DSPAalpha1, respectively. Our results show that for both activators the products released during fibrinolysis are very effective cofactors for plasminogen activation, and both t-PA and DSPAalpha1 cofactor activity are strongly down-regulated by TAFIa.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.