Circulating mitochondrial stress 70 protein/mortalin and cytosolic Hsp70 in blood: Risk indicators in colorectal cancer.

Mitochondrial mortalin and cytosolic Hsp70 are essential chaperones overexpressed in cancer cells. Our goals were to reproduce our earlier findings of elevated circulating levels of mortalin and Hsp70 in colorectal cancer (CRC) patients with a larger patient cohort, to compare death risk assessment of mortalin, Hsp70, CEA and C19-9 and to assess their prognostic value in various CRC stages. Mortalin, Hsp70, CEA and CA19-9 levels were determined in sera of 235 CRC patients enrolled in the study and followed up 5 years after surgery. Association between their concentrations and patients' survival was analyzed by Kaplan-Meier estimator and subjected to Cox Proportional hazards analysis. Serum level of mortalin was independent of that of Hsp70, CEA and CA19-9, whereas Hsp70 level weakly correlated with CEA and CA19-9 levels. Improved short-term survival was found in early or advanced disease stages associated with lower mortalin and Hsp70 levels. Cox regression analysis showed a high mortality hazard (HR = 3.7, p < 0.001) in patients with both high mortalin and Hsp70 circulating levels. Multivariate analysis showed that high mortalin and Hsp70 significantly enhances risk score over a baseline model of age, number of affected lymph nodes, CEA, CA19-9, disease stage and perioperative therapy. Analysis of mortalin and Hsp70 in CRC patients' sera adds a high prognostic value to TNM stage and to CEA and CA19-9 and identifies patients with lower or higher survival probability in all CRC stages. Determination of mortalin and Hsp70 in blood could be a useful additive prognostic tool in guiding clinical management of patients.

Metastasis blood test by flow cytometry: in vivo cancer spheroids and the role of hypoxia.

Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis-compared to the high circulating tumor-cell numbers (CTCs)-appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid-size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light-scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC-association with in vivo spheroids. We propose that the mechanism involves a portable "micro-niche" in spheroids that may support CSC-survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression.

Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin.

By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin-trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1M ratio with Ki values of 2.8 × 10(-12)M and 1.87 × 10(-12)M, respectively. The P1-P1' residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Internal friction in enzyme reactions.

The empirical concept of internal friction was introduced 20 years ago. This review summarizes the results of experimental and theoretical studies that help to uncover the nature of internal friction. After the history of the concept, we describe the experimental challenges in measuring and interpreting internal friction based on the viscosity dependence of enzyme reactions. We also present speculations about the structural background of this viscosity dependence. Finally, some models about the relationship between the energy landscape and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions are also discussed.

[Surgery of peritoneal surface malignancies - surgical cytoreduction and hyperthermic intraperitoneal chemotherapy]. / A peritoneális felszín tumorainak sebészete ­ citoreduktív sebészet és hipertermiás intraperitoneális kemoterápia.

Peritoneal carcinosis has historically been considered as inoperable, although the technique of its resesection together with high dose intraperitoneal chemotherapy potentiated by heat has been described decades ago. It has not became a widely practiced routine except in specialized centers - the complex technique, weakly standardized but resource demanding chemotherapy, lacking financial background and the many times questionable clinical benefit at a cost of high surgical load might have been the key factors. Refined technology, developing chemotherapy protocols together with growing clinical evidence are now more sharply delineating the range of indications where the procedure might be beneficial, increases survival, or is the only curative therapy. These include tumors of the appendix and pseudomyxoma peritonei, mesothelioma, and selected cases of ovarian, colorectal and gastric cancer. In addition to technical description of the intervention, we summarize the currently valid indications and describe our institutional protocol for the treatment of appendiceal malignancies.

Serum fetuin-A is decreased in cirrhotic patients with Wilson's disease.

INTRODUCTION: Wilson's disease may lead to cirrhosis, but timely medical treatment could slow down its progression. Clinical markers helping early diagnosis are essential. Decreased fetuin-A concentration has been reported in cirrhosis of different etiologies. The aim of this study was to investigate whether decreased serum fetuin-A concentration could identify patients with Wilson's disease who developed cirrhosis. MATERIALS AND METHODS: In this cross-sectional study we determined the serum fetuin-A concentration of 50 patients with Wilson's disease. We analyzed the data of patients with liver involvement, comparing cirrhotic and non-cirrhotic patients. RESULTS: Among patients with liver involvement those with cirrhosis had significantly lower fetuin-A and albumin level, white blood cell and platelet count. Fetuin-A negatively correlated with disease duration, bilirubin level, positively with total protein and albumin concentration, but not with copper and ceruloplasmin concentrations or markers of systemic inflammation. In multivariate analysis with fetuin-A and the Nazer score or its parameters only fetuin-A was a significant determinant of having cirrhosis. In receiver operator curve analysis among patients with liver involvement the fetuin-A level of 523 µg/ml was associated with cirrhosis with 82% sensitivity and 87% specificity. The presence of the H1069Q mutation was not associated with alteration in fetuin-A concentration. CONCLUSIONS: The serum concentration of fetuin-A is a sensitive marker of liver cirrhosis in Wilson's disease, independently of the H1069Q mutation, ceruloplasmin concentration or systemic inflammation.

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action.

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O(δ2)-C(γ) bond appears to be a double bond, with O(δ2) involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O(δ1) atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF(obs) - DF(calc) density above 2.5 σ next to O(δ1). As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK(a) of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK(a) histidine theory.

Temperature dependence of internal friction in enzyme reactions.

Our aim was to elucidate the physical background of internal friction of enzyme reactions by investigating the temperature dependence of internal viscosity. By rapid transient kinetic methods, we directly measured the rate constant of trypsin 4 activation, which is an interdomain conformational rearrangement, as a function of temperature and solvent viscosity. We found that the apparent internal viscosity shows an Arrhenius-like temperature dependence, which can be characterized by the activation energy of internal friction. Glycine and alanine mutations were introduced at a single position of the hinge of the interdomain region to evaluate how the flexibility of the hinge affects internal friction. We found that the apparent activation energies of the conformational change and the internal friction are interconvertible parameters depending on the protein flexibility. The more flexible a protein was, the greater proportion of the total activation energy of the reaction was observed as the apparent activation energy of internal friction. Based on the coupling of the internal and external movements of the protein during its conformational change, we constructed a model that quantitatively relates activation energy, internal friction, and protein flexibility.

Reversible heat-induced dissociation of ß2-microglobulin amyloid fibrils.

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of ß(2)-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized ß2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of ß2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of ß2m fibril solution. 0.5 mM SDS completely prevented ß2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.

Serum fetuin-A in metabolic and inflammatory pathways in patients with myocardial infarction.

BACKGROUND: Although multifunctional glycoprotein α2HS-glycoprotein/human fetuin-A (AHSG) is involved in atherosclerosis, it is not clear whether high or low concentrations are more important. We studied the correlation of serum AHSG with adiponectin, leptin, resistin, C-reactive protein (CRP) and tumour necrosis factor-α (TNF-α) to see whether its metabolic effects or its involvement in subclinical inflammation are dominant in patients with established coronary disease. MATERIALS AND METHODS: In this cross-sectional study, AHSG concentration was determined in sera of 171 patients (age: 62 ± 6 years, mean ± SD) with previous myocardial STEMI infarction and normal renal function and 81 age-matched healthy controls by radial immunodiffusion. Results Patients had increased AHSG levels (673 ± 103 vs. 619 ± 96 mg L(-1), P < 0·001) compared to controls. Obese and diabetic patients had higher AHSG concentration than those with normal BMI or without diabetes but even the latter group had elevated AHSG levels (667 ± 101 mg L(-1), n = 88) compared to controls (P = 0·002). Serum AHSG correlated negatively with adiponectin (r = -0·236, P = 0·006) even after adjusting for BMI (r = -0·177, P = 0·043). AHSG determined adiponectin levels independently from BMI, age and other adipocytokines (P = 0·014). The correlation between leptin and AHSG (r = 0·321, P = 0·021) weakened following adjustment for BMI (r = 0·209, P = 0·072). Serum AHSG did not correlate significantly with CRP, resistin and TNF-α concentrations. BMI and resistin but not AHSG determined TNF-α levels independently. CONCLUSIONS: AHSG is elevated in sera of patients with previous myocardial infarction. Association with adipokines favours the concept that AHSG is involved in atherosclerosis more likely through metabolic pathways (insulin resistance, obesity and adipocyte dysfunction) than by inflammation in patients with post-myocardial infarction.

Anoxia leads to a rapid translocation of human trypsinogen 4 to the plasma membrane of cultured astrocytes.

Trypsinogen 4 is specifically expressed in the human brain, mainly by astroglial cells. Although its exact role in the nervous tissue is yet unclear, trypsin 4-mediated pathological processes were suggested in Alzheimer's disease, multiple sclerosis and ischemic injury. In the present study, we analyzed the intracellular distribution of fluorescently tagged human trypsinogen 4 isoforms during normal and anoxic conditions in transfected mouse primary astrocytes. Our results show that initiation of anoxic milieu by the combined action of KCN treatment and glucose deprivation rapidly leads to the association of leader peptide containing trypsinogen 4 constructs to the plasma membrane. Using rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide), a synthetic chromogen peptide substrate of trypsin, we show that anoxia can promote extracellular activation of trypsinogen 4 indicating that extracellular activation of human trypsinogen 4 can be an important component in neuropathological changes of the injured human brain.

Mechanism of lysophosphatidic acid-induced amyloid fibril formation of beta(2)-microglobulin in vitro under physiological conditions.

Beta(2)-microglobulin- (beta2m-) based fibril deposition is the key symptom in dialysis-related amyloidosis. beta2m readily forms amyloid fibrils in vitro at pH 2.5. However, it is not well understood which factors promote this process in vivo, because beta2m cannot polymerize at neutral pH without additives even at elevated concentration. Here we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator, promotes amyloid formation under physiological conditions through a complex mechanism. In the presence of LPA, at and above its critical micelle concentration, native beta2m became sensitive to limited proteolytic digestion, indicating increased conformational flexibility. Isothermal titration calorimetry indicates that beta2m exhibits high affinity for LPA. Fluorescence and CD spectroscopy, as well as calorimetry, showed that LPA destabilizes the structure of monomeric beta2m inducing a partially unfolded form. This intermediate is capable of fibril extension in a nucleation-dependent manner. Our findings also indicate that the molecular organization of fibrils formed under physiological conditions differs from that of fibrils formed at pH 2.5. Fibrils grown in the presence of LPA depolymerize very slowly in the absence of LPA; moreover, LPA stabilizes the fibrils even below its critical micelle concentration. Neither the amyloidogenic nor the fibril-stabilizing effects of LPA were mimicked by its structural and functional lysophospholipid analogues, showing its selectivity. On the basis of our findings and the observed increase in blood LPA levels in dialysis patients, we suggest that the interaction of LPA with beta2m might contribute to the pathomechanism of dialysis-related amyloidosis.

Revisiting the mechanism of the autoactivation of the complement protease C1r in the C1 complex: structure of the active catalytic region of C1r.

C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control protein (CCP) modules. The activated CCP1-CCP2-SP fragment makes up a dimer in a head-to-tail fashion similarly to the previously characterized zymogen. The present structure shows an increased number of stabilizing interactions. Moreover, in the crystal lattice there is an enzyme-product relationship between the C1r molecules of neighboring dimers. This enzyme-product complex exhibits the crucial S1-P1 salt bridge between Asp631 and Arg446 residues, and intermolecular interaction between the CCP2 module and the SP domain. Based on these novel structural information we propose a new split-and-reassembly model for the autoactivation of the C1r. This model is consistent with experimental results that have not been explained adequately by previous models. It allows autoactivation of C1r without large-scale, directed movement of C1q arms. The model is concordant with the stability of the C1 complex during activation of the next complement components.

When the surface tells what lies beneath: combinatorial phage-display mutagenesis reveals complex networks of surface-core interactions in the pacifastin protease inhibitor family.

Pacifastin protease inhibitors are small cysteine-rich motifs of approximately 35 residues that were discovered in arthropods. The family is divided into two related groups on the basis of the composition of their minimalist inner core. In group I, the core is governed by a Lys10-Trp26 interaction, while in group II it is organized around Phe10. Group I inhibitors exhibit intriguing taxon specificity: potent arthropod-trypsin inhibitors from this group are almost inactive against vertebrate enzymes. The group I member SGPI-1 and the group II member SGPI-2 are extensively studied inhibitors. SGPI-1 is taxon-selective, while SGPI-2 is not. Individual mutations failed to explain the causes underlying this difference. We deciphered this phenomenon using comprehensive combinatorial mutagenesis and phage display. We produced a complete chimeric SGPI-1 / SGPI-2 inhibitor-phage library, in which the two sequences were shuffled at the highest possible resolution of individual residues. The library was selected for binding to bovine trypsin and crayfish trypsin. Sequence analysis of the selectants revealed that taxon specificity is due to an intra-molecular functional coupling between a surface loop and the Lys10-Trp26 core. Five SGPI-2 surface residues transplanted into SGPI-1 resulted in a variant that retained the "taxon-specific" core, but potently inhibited both vertebrate and arthropod enzymes. An additional rational point mutation resulted in a picomolar inhibitor of both trypsins. Our results challenge the generally accepted view that surface residues are the exclusive source of selectivity for canonical inhibitors. Moreover, we provide important insights into general principles underlying the structure-function properties of small disulfide-rich polypeptides, molecules that exist at the borderline between peptides and proteins.

The crystal structure of a trypsin-like mutant chymotrypsin: the role of position 226 in the activity and specificity of S189D chymotrypsin.

The crystal structure of the S189D+A226G rat chymotrypsin-B mutant has been determined at 2.2 angstroms resolution. This mutant is the most trypsin-like mutant so far in the line of chymotrypsin-to-trypsin conversions, aiming for a more complete understanding of the structural basis of substrate specificity in pancreatic serine proteases. A226G caused significant rearrangements relative to S189D chymotrypsin, allowing an internal conformation of Asp189 which is close to that in trypsin. Serious distortions remain, however, in the activation domain, including zymogen-like features. The pH-profile of activity suggests that the conformation of the S1-site of the mutant is influenced also by the P1 residue of the substrate.

High serum Hsp70 level predicts poor survival in colorectal cancer: Results obtained in an independent validation cohort.

BACKGROUND: Hsp70 plays important role in the development and progression of cancer. Previously we described the association between serum Hsp70 levels and mortality of colorectal cancer. OBJECTIVE: In this new prospective study we aimed to confirm and extend our previous findings in a larger cohort of patients, based on a longer follow-up period. METHODS: Two hundred and thirty-two patients diagnosed with colorectal cancer were enrolled in the study. Baseline serum Hsp70 level and classical biomarker levels were measured. Patients were treated according to stage of the tumor and follow-up lasted for a median 46.4 months. RESULTS: We found that serum Hsp70 concentrations increase significantly with stage of the disease (1.79; 2.23 and 3.21 ng/ml in stage I+II, III and IV respectively, p= 0.012 and 0.002, Mann-Whitney test) and with other known biomarkers of the disease. We managed to confirm our previous findings that high baseline serum Hsp70 level (> 1.64 ng/ml) predicted poor 5-year survival (risk of death HR: 1.94 CI: 1.294-2.909; univariate; HR: 2.418 CI: 1.373-4.258; multivariate Cox regression analysis) in the whole patient population and also in subgroups of stage IV and stage III disease. The strongest association was observed in women under age of 70 (HR: 8.12, CI: 2.02-35.84; p= 0.004; multivariate Cox regression). The power of this colorectal cancer prognostic model could be amplified by combining Hsp70 levels and inflammatory markers. Patients with high Hsp70, CRP and high baseline WBC or platelet count had 5-times higher risk of death (HR: 5.07 CI: 2.74-9.39, p< 0.0001; and HR: 4.98 CI: 3.08-8.06, p< 0.0001 respectively). CONCLUSIONS: These results confirm and validate our previous findings that serum Hsp70 is a useful biomarker of colorectal cancer.

Platelet Count, ADAMTS13 Activity, von Willebrand Factor Level and Survival in Patients with Colorectal Cancer: 5-Year Follow-up Study.

Distant metastasis is a major cause of colorectal cancer-related death, but the mechanism of tumour progression is not fully understood. There is growing evidence of an interaction between tumour cells and platelets which may influence tumour progression and metastasis formation. Quality and quantity of von Willebrand factor may regulate the interaction between tumour cells and platelets. Our aim was to measure the platelet count, von Willebrand factor antigen (VWF:Ag) levels and ADAMTS13 activity in a large (n = 232) cohort of colorectal cancer patients and to examine their relationships with the stage of the disease and 5-year survival without thrombotic complications using multivariable models. Significantly higher platelet counts (p = 0.005), VWF:Ag levels (p = 0.008) and decreased ADAMTS13 activity (p = 0.006) were observed in patients with metastatic disease. Results of the Kaplan-Meier analysis showed that lower platelet counts (p < 0.0001), lower VWF:Ag (p = 0.0008) levels and higher ADAMTS13 activity (p < 0.0001) were associated with better event-free survival. Finally, to investigate the association between overall event-free survival and the three study variables, multivariate Cox proportional hazard models were generated. All models were adjusted for age, gender and disease stage. Platelet count, ADAMTS13 activity or VWF:Ag level were incorporated and all of these variables turned out to be age-, gender- and stage-independent predictors of mortality (all hazard ratio >1.7, p < 0.05). In summary, this is the first observational study reporting association between higher mortality or thrombotic complications and increased platelet count, increased VWF:Ag levels and decreased ADAMTS13 activity in colorectal cancer.

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: role of local internal viscosity in protein dynamics.

Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5-60 degrees C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region.

Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate.

The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.