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.

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.