Recent trends in protease-catalyzed peptide synthesis.

Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

Use of liquid chromatography-mass spectrometry coupling for monitoring the serralysin-catalyzed hydrolysis of a peptide library.

The use of a peptide library of limited size, is considered to be more appropriate for studying a protease with a complex specificity, but very sensitive and efficient analytical techniques must be used. We have designed and synthesized a 49-peptide library of the type Z-AlaXXAla(amide) (X=Ala, Leu, Val, Phe, Ser, Arg, Glu) for studying the Pseudomonas aeruginosa serralysin specificity. All compounds of the peptide library could be identified by a LC-MS procedure. After hydrolysis of the library by pseudomonal serralysin, the LC-MS procedure also allowed the identification of the hydrolysis products and the different cleavage sites of the substrates.

Use of a 49-peptide library for a qualitative and quantitative determination of pseudomonal serralysin specificity.

The Pseudomonas aeruginosa serralysin (E.C. 3.4.24.40.), which is a zinc-dependent metalloprotease from the metzincin superfamily, has quite a broad specificity, which has not yet been clearly identified. We have studied it with an original approach, using a 49-peptide library of the type Z-AXXA (amide) (X = A, L, V, F, S, R, E). The library was analyzed by LC-MS before and after enzymatic hydrolysis. A great number of hydrolyzed peptides were screened and the preferential hydrolysis was the X-X peptide bond, even if in some cases, A-X and X-A bond could be hydrolyzed. No amino acids with a ionized side chain could be found in the P1' position. The results obtained suggest that the specificity in the P1' position, where an hydrophobic residue was preferentially found, seems more selective that in the Pn position. The P1 position was not very specific, but, on a quantitative point of view, the enzymatic activity was particularly increased when R, F or A were in this position. The results allow us to define the P1' and P1 residues for an optimal substrate of pseudomonal serralysin and usable for the design and the synthesis of a specific inhibitor.

Specificity of Pseudomonas aeruginosa serralysin revisited, using biologically active peptides as substrates.

The characterization of the specificity of alkaline protease from Pseudomonas aeruginosa has not yet been clearly defined. Some previous results suggested that its specificity was influenced more by amino acids far from the hydrolyzed peptide bond, than by amino acids in P1 or P'1 position. From other data, it was a C-(COOH)-type endoprotease where the preferential amino acid in P1 position was an arginine residue. We have studied the hydrolysis of several biologically active peptides. Many various sites of cleavage have been characterized but no arginine in P1 position was found, despite the presence of arginine in the peptide sequence. In fact P1 and P'1 position could be occupied by various amino acids. It seems unlikely that Pseudomonas alkaline protease may only be considered as a protease specific to arginine in P1 position. On the other hand, we have observed that increase of the peptide chain length led to an important increase of the hydrolysis rate, suggesting an extended number of subsites.

Hydrophobic interactions are involved in the inhibition of human leukocyte elastase by alkyltrimethylammonium salts.

Electrostatic forces and hydrophobic interactions had been suggested to modify the adsorption of elastases onto insoluble fibrous elastin, which is the initial stage of elastolysis, but conflicting results had been obtained, and comparison between compounds with different structures was difficult. In order to explore these observations, we have studied the effect of six alkyltrimethylammonium bromides, with alkyl chain length ranging from six to 16 carbon atoms, on human leucocyte elastase activities, either with a synthetic substrate or with insoluble elastin. The enzymatic studies were performed either spectrophotometrically or using conductimetry, and direct binding on to elastin was conductimetrically measured. Binding of the alkyltrimethylammonium salts is increasing with alkyl chain length and we could demonstrate a cooperative binding for tetra- and hexadecyl chains. No effect of the six compounds could be evidenced on hydrolysis of a specific synthetic substrate. With insoluble elastin, elastolysis inhibition could be demonstrated for alkyl chain longer than ten carbon atoms, the effect increasing with chain length. A similar inhibition was observed with the soluble kappa-elastin, but it was less effective. The study shows that the interaction between the alkyltrimethylammonium salts and elastin plays a major role in the inhibitory potency of these molecules. As this effect is enhanced with alkyl chain length, it was concluded that hydrophobic interactions favour their binding, protecting elastin against elastase adsorption.

Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.

The effects of albuterol on power output in non-asthmatic athletes.

The purpose of this study was to evaluate the effect of the beta 2-agonist albuterol (salbutamol) at twice the normal dosage (360 micrograms) on power output during a 30-second Wingate test and pulmonary function in highly trained cyclists (4 category 1 and 10 category II U.S.C.F. track cyclists). The cyclists did not have a history of exercise induced bronchial spasms, and a 5 step methacholine challenge confirmed all subjects to be non-asthmatic. The project was performed in a random block, double blind design. Twenty minutes before the 30-second Wingate cycle ergometer exercise, albuterol (90 micrograms per dose) or a saline placebo was administered by inhaler in 4 metered doses. Pulmonary function tests were performed at rest, 20 minutes post-inhalation, and 5, 10, 15 minutes post-exercise. After a standard warm-up, a 30-second Wingate anaerobic power test was performed on a cycle ergometer at a resistance of 0.10 kg (kg body mass)-1. Multi-variate ANOVA revealed no significant difference between the albuterol and placebo treatment for the anaerobic power measures: peak power (1,136.7 +/- 40.9 vs 1,124.8 +/- 39.8 W, mean +/- s.e.), total work (27,213.6 +/- 653.1 vs 27,093.3 +/- 677.4j), time to peak power (4.5 +/- 0.2 vs 4.8 +/- 0.5 s), and fatigue index (16.5 +/- 1.8 vs 16.6 +/- 1.8 W.s-1). Peak heart rate (181.6 +/- 3.7 vs 181.4 +/- 3.8 bpm), or blood lactate (14.0 +/- 0.9 vs 13.8 +/- 0.8 mmol.l-1) 3 min after the exercise bout were not significantly different between treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute inhalation of albuterol on submaximal and maximal VO2 and blood lactate.

The acute effects of inhaled albuterol, a selective beta-2 adrenergic agonist, on measures of endurance cycling performance and pulmonary function were assessed in 21 competitive road cyclists. A 5 step methacholine challenge revealed all cyclists to be non-asthmatic. Albuterol (A) total dose 360 micrograms or a saline placebo (P) was administered by inhaler, in 4 metered doses of 90 micrograms each, 15 minutes before cycle ergometry exercise. Heart rate, whole blood lactate, perceived exertion and VO2 were determined at the submaximal workloads of 150, 200, 225, 250, 275, 300 watts and at max. Pulmonary function tests determining forced vital capacity, forced expiratory volume during the first second of expiration, forced mid-expiratory flow and maximal voluntary ventilation were performed prior to and 10 minutes after inhalation; and 5, 10 and 15 minutes after termination of the exercise protocol. Heart rate was significantly greater during the A compared to the P treatment at 200 (150.8 +/- 2.5 vs 146.7 +/- 2.8 beats per minute), 225 (159.7 +/- 2.4 vs 154.6 +/- 2.7 beats per minute) and 250 watts (166.9 +/- 2.4 vs 164.4 +/- 2.6 beats per minute). Whole blood lactate was significantly greater during the A compared to the P treatment at 275 watts (4.7 +/- 0.3 vs 4.2 +/- 0.4 mmol.l-1). No other significant differences were found between the 2 treatments at any time point. These data indicate that the acute effect of albuterol inhalation at twice the recommended dosage has no positive effect on endurance performance measures or pulmonary function in athletes who are not asthmatic.

Fifteen years of amateur boxing injuries/illnesses at the United States olympic training center.

We examined the incidence of health problems in elite-level amateur boxing athletes who sparred, trained, or competed at the United States Olympic Training Center in Colorado Springs, Colorado from January 1, 1977 through June 30, 1992. We think this is the first study to examine both injuries and illnesses in a population of elite-level athletes. We collected data on 1,776 reported problems (1219 injuries, 557 illnesses) from standard medical report forms completed by the permanent and volunteer sports medicine staff. We classified the information based on type, body region, location, description, and occurrence. There were significant differences between the frequency of injuries and illnesses and between the classifications and regions for each type of problem. Collectively, serious injuries represented only a relatively small percentage (6.1%) of all problems. We concluded that illnesses comprised a small but important portion of problems, that most illnesses involved respiratory tract infections (71%), that there is only a small risk for serious injury, and that injuries occur in a hierarchy of upper extremity (441, 25%), head/face (344, 19%), lower extremity (267, 15%), and spinal column (167, 9%) for amateur boxers.

Comparison of four procedures for measuring elastase production by Pseudomonas aeruginosa strains from cystic fibrosis patients.

Forty-five Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. The elastase production of each strain was assayed in the culture supernatant using four different procedures, i.e. two immunological assays (RIA and ELISA), and two enzymatic assays, the latter employing either elastin or tetraalanine as substrate, with conductometric measurement of substrate hydrolysis. Elastase concentrations were determined from standard curves prepared with the same purified elastase, and expressed in mg of elastase per litre of supernatant. The resulting values were in the range reported in the literature, and differed greatly from one strain to another (0-230 mg/l). Linear relationships were found when assays were compared in pairs. Significant correlation coefficients were obtained (r greater than 0.76, p less than 0.001) but the values were quite different for different assays. Thus, ELISA measurements were always from three to five times higher, and RIA results were from two to five times lower, than those from the other assays. Enzymatic assays with elastin gave higher values than those using tetraalanine. Most P. aeruginosa strains produce two other proteinases, alkaline proteinase and Las A protein. Both enzymes have limited elastolytic and peptidasic activities. The presence of alkaline proteinase does not result in falsely elevated elastase values, but an increase of elastase activity was observed when Las A was preincubated with elastin. Since this increase was not observed when tetraalanine was used as the substrate, the presence of Las A in the supernatants could explain the differences observed between the enzymatic assays. The assay with the synthetic substrate is therefore preferred.

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity.

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.

Age-dependent variations of human PMNL elastase activity as a function of pH, ionic strength and calcium concentration.

Human leukocyte elastase (HLE) has been implicated in the pathomechanism of various diseases, such as emphysema and atherosclerosis. The incidence of these diseases is increasing with aging. Therefore, it can be supposed that the HLE activity is changing with aging according to the well known age-related physiological alterations. Thus, the effects of pH, NaCl and calcium concentrations on HLE activity, separated from polymorphonculear leucocytes (PMNLs) of healthy, young (<35 years) and elderly (>75 years) subjects, were studied by measuring the activity with synthetic substrate and with bovine and human atherosclerotic and non-atherosclerotic aortic elastin. From our results, it may be concluded, that the elastolytic activity of HLE separated from PMNLs of elderly subjects is more sensitive to ionic strength, to pH and to the calcium concentration of the medium, than the HLE activity of young subjects. The elastolytic activity of HLE, of both young and old subjects, increases dramatically on atherosclerotic aortic elastin in the presence of calcium. These findings might explain, at least partly, the increased incidence of atherosclerosis with aging.

Human aortic elastin from normal individuals and atherosclerotic patients: lipid and cation contents; susceptibility to elastolysis.

Aortic elastins, isolated from 30 humans of different ages, were purified by alkaline extraction, and separated into two groups depending on the presence of atherosclerotic plaques and calcification (grades 0 and 1). It was confirmed that the severity of atherosclerosis increases significantly with age (P less than 0.001) and elastin content decreases with atherosclerosis (P less than 0.001). The hydrolysis of the aortic elastins using pancreatic porcine elastase (PPE) was studied. It was observed that increased elastolytic activities are connected with severity of atherosclerosis (P less than 0.001) and both Vm and Km apparent kinetic parameters are affected (P less than 0.001). Correlation tests have shown that enzymatic hydrolysis is significantly modified by cholesterol (P less than 0.05), calcium (P less than 0.001) and magnesium concentrations (P less than 0.01) but only cholesterol changes significantly Vm and Km parameters.

Hydrolysis of alanine oligomers and of elastin by P. aeruginosa proteinases and thermolysin.

The hydrolysis of alanine oligomers by P. aeruginosa proteinases, thermolysin and porcine pancreatic elastase was studied. The concentrations of substrates and cleavage products were determined using reverse phase high pressure liquid chromatography. Tetraalanine was the shortest oligomer for which we could demonstrate hydrolysis by all the proteinases, except for porcine pancreatic elastase which only significantly hydrolyzed peptides longer than hexaalanine. Porcine pancreatic elastase hydrolyzes hexaalanine at a single site, whereas the other enzymes may split it either into two trialanine molecules, or into di- and tetraalanine, the latter being further cleavable to dialanine. A kinetic model based on first-order kinetic rate constants is proposed and the individual constants determined. Although P. aeruginosa elastase and thermolysin are closely similar in structure, they have shown a marked difference in their hydrolysis of either elastin or tetraalanine. Elastolytic activity of thermolysin was higher than that of elastase but tetraalanine was hydrolyzed more slowly by thermolysin.

[Mechanism of action of proteinases of Pseudomonas aeruginosa]. / Mécanisme d'action des protéinases de Pseudomonas aeruginosa.

Of the two proteases produced by Pseudomonas aeruginosa, one whose optimal pH is neutral, exhibits elastolytic activity. This elastase is produced as a prepropeptide, subsequently modified by proteolysis, and finally secreted as an active enzyme. Both proteases act mainly on hydrophobic aminoacids. The most important site for the elastase seems to be the P'1 site where Phe, Tyr and Leu residues enhance hydrolysis. The alkaline protease is less specific of this site. At least four aminoacids are needed to obtain measurable rates of hydrolysis. A synthetic substrate, Ala-Ala-Phe-Ala, is proposed for conductimetric measurements of Pseudomonas aeruginosa elastase activities in the nanomolar range. A review of recent studies shows that a very wide range of proteins can be hydrolyzed by the two Pseudomonas aeruginosa proteases, a fact which may explain why these enzymes are major determinants of the bacteria's infectivity.

Elastolysis of kappa-elastin sub-fractions by pancreatic and leukocyte elastases.

Kappa-elastin peptides, obtained from insoluble elastin by organo-alkaline hydrolysis, were fractioned by gel filtration on Biogel agarose. Rates of hydrolysis by pancreatic and leukocyte elastases of the fractions were measured using a conductometric method. Kinetics obey to Michaelis-Menten model for both substrates and enzymes. KM and Vmax values derived from Lineweaver-Burk plots indicate that, if KM remains quite constant, differences were observed in catalytic rates. The kcat values decreased with molecular-weight, the high-molecular-weight kappa-elastin peptides being hydrolyzed 3 to 5 times faster than the low-molecular-weight ones. Apparent differences between potentiometric (pH-Stat) and conductometric results were discussed, in relation with buffer capacity of soluble and insoluble elastins.

Elastolytic activity of Pseudomonas aeruginosa elastase.

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.

Automated conductimetric assay of human serum cholinesterase activity.

Serum cholinesterase activity was determined by conductimetry using samples in the microliter range. Butyrylcholine iodide was demonstrated to be a convenient substrate for the conductimetric assay. Validation of the microassay was made by using either purified enzyme or control serum. In the range of 0-60 U/l, a linear relationship was demonstrated. Correlation with a reference spectrophotometric method was obtained with a slope of 1.18. An explanation of this value is proposed, as different hydrolysis rates were obtained with human sera, depending on the substrate used (butyrylthio- or butyryl-choline ester).

Elastolysis of normal and partially cross-linked elastin.

Rate of elastolysis by pancreatic and leukocyte elastases of normal and copper deficient porcine aortic elastins were measured using a conductimetric method. Kinetics obey to Michaelis-Menten model for both substrates and enzymes. KM and Vmax values derived from Lineweaver-Burk plots indicate that, if a near uniformity exists in KM, differences were observed in catalytic rates, kcat increasing approximately 40% for copper deficient elastin elastolysis by leukocyte elastase. This higher susceptibility to proteolysis may have implications for understanding turnover of elastin in tissues.