Lung 11990) 168:323-332
Lung
icl Springer-Verlag New York Inc. 1990
Inhibition of the Activity of Human Leukocyte Elastase by Lipids Particularly Oleic Acid and Retinoic Acid David Skhm, Revital Rappaport, and Micha Vered F:aCtllly of Agriculture, Hebrew University, Rehovot, Israel
Abstract. The effect of various natural hydrophobic lipids on the in vitro and in vivo activity of human leukocyte elastase has been examined. In vitro studies using 2 different substrates indicated that fatty acids inhibit human leukocyte elastase activity, with maximum inhibition observed with oleic acid. Triolein, cholesterol, and B-carotene caused little inhibition. The presence of a carboxyl group appears important since retinoic acid but not retinol also inhibited activity. In vivo studies of an emphysema model in mice indicated that intrapulmonary instillation of oleic or retinoic acid reduced lung injury caused by human leukocyte elastase. The possibility of using these compounds to diminish elastolytic damage in emphysema is raised. Key words: Elastase--Emphysema--Lipids--Oleic acid--Retinoic acid. Introduction Human leukocyte elastase (HLE) is a basic protein of M,. 30000, that has been implicated as the principal destructive agent of lung connective tissue in pulmonary emphysema [10]. HLE is one of the serine protease enzymes that have been extensively characterized mechanistically [23]. These proteases catalyze a series of hydrolytic reactions involving the reversible formation of an encounter complex between enzyme and substrate, followed by production and subsequent hydrolysis of an acyl-enzyme intermediate [20]. HLE uses general acid-base catalysis to stabilize transition states for ester and amide hydrolysis. Substrate structure is thus critical for involvement in the rate-limiting processes [21 ]. Several differAddress o/Jprint requests to: Dr. D. Sklan, Faculty of Agriculture, PO Box 12. Rehovot 76I00, Israel
324
1). Skhin cl al.
ent synthetic substrales have been used to determine HLE activity, mainly peptide-p-nitroanilides. However, differences in water solubility and hydrophobicity have resulted in somewhat differing results with different substrates [4, 7]. It has been reported that tetrapeptides containing oleic acid inhibit H LE activity [9]. In addition oleic acid (OA) was shown to inhibit human granulocyte elastase activity towards BOC-ala-ala-pro-ala-p-NA (BOC, [I ]) and towards Nmethoxysuccinyl-(alanyl)2-pro-val-p-nitroanilide [6], whereas the corresponding alcohols enhanced activity slightly [I]. Thus it appears that fatty acids influence HLE activity. The objective of this study was to determine the effect of various na|urally occurring hydrophobic materials on the activity of H LE towards 2 substrates, Nsuccinyl-(alanyl)3-p-nitroanilide (SAAAPN) and N-methoxysuccinyl-(ahmyl)~_pro-val-p-nitroanilide (MeSAAPVPN) in vitro and to determine their effects on an emphysema model in rive.
Materials and Methods FILE was purchased from Elastin Products Inc. (Pacific, M e ) ~zt-antitrypsin inhibitor, c h o n d r o i t i n 'qllfate, and lipids were from Sigma Chemical Co. (MO), 13-cis retinoic acid was a gift f r o m I ltffl'mann-t,a Roche (Nutty, N J), In kinetic experimcnls with 2 synthetic nilroaniline peptide substrates ( S A A A P N ,:.md t'Vle,';,,kAIWPN. Sigma Chemical Co.. Me)). the reaction rate was determined in a U v i k o n ~,pectrophotometer using a modification o f the method o f Bieth el al. 14t in which the rate o f p-nitroaniline l\)rmation is measured. To a disposable cuvette containing 0.9 ml o f 0.01 M lris-llCt. 0.15 M NaCI, pH g.0. 0, I ml lipids or liposome solulions in buffer were added to the desired linal concentration and 0.06 /zM enzyme added and preincubated for 5 rain tit 37
Lung
icl Springer-Verlag New York Inc. 1990
Inhibition of the Activity of Human Leukocyte Elastase by Lipids Particularly Oleic Acid and Retinoic Acid David Skhm, Revital Rappaport, and Micha Vered F:aCtllly of Agriculture, Hebrew University, Rehovot, Israel
Abstract. The effect of various natural hydrophobic lipids on the in vitro and in vivo activity of human leukocyte elastase has been examined. In vitro studies using 2 different substrates indicated that fatty acids inhibit human leukocyte elastase activity, with maximum inhibition observed with oleic acid. Triolein, cholesterol, and B-carotene caused little inhibition. The presence of a carboxyl group appears important since retinoic acid but not retinol also inhibited activity. In vivo studies of an emphysema model in mice indicated that intrapulmonary instillation of oleic or retinoic acid reduced lung injury caused by human leukocyte elastase. The possibility of using these compounds to diminish elastolytic damage in emphysema is raised. Key words: Elastase--Emphysema--Lipids--Oleic acid--Retinoic acid. Introduction Human leukocyte elastase (HLE) is a basic protein of M,. 30000, that has been implicated as the principal destructive agent of lung connective tissue in pulmonary emphysema [10]. HLE is one of the serine protease enzymes that have been extensively characterized mechanistically [23]. These proteases catalyze a series of hydrolytic reactions involving the reversible formation of an encounter complex between enzyme and substrate, followed by production and subsequent hydrolysis of an acyl-enzyme intermediate [20]. HLE uses general acid-base catalysis to stabilize transition states for ester and amide hydrolysis. Substrate structure is thus critical for involvement in the rate-limiting processes [21 ]. Several differAddress o/Jprint requests to: Dr. D. Sklan, Faculty of Agriculture, PO Box 12. Rehovot 76I00, Israel
324
1). Skhin cl al.
ent synthetic substrales have been used to determine HLE activity, mainly peptide-p-nitroanilides. However, differences in water solubility and hydrophobicity have resulted in somewhat differing results with different substrates [4, 7]. It has been reported that tetrapeptides containing oleic acid inhibit H LE activity [9]. In addition oleic acid (OA) was shown to inhibit human granulocyte elastase activity towards BOC-ala-ala-pro-ala-p-NA (BOC, [I ]) and towards Nmethoxysuccinyl-(alanyl)2-pro-val-p-nitroanilide [6], whereas the corresponding alcohols enhanced activity slightly [I]. Thus it appears that fatty acids influence HLE activity. The objective of this study was to determine the effect of various na|urally occurring hydrophobic materials on the activity of H LE towards 2 substrates, Nsuccinyl-(alanyl)3-p-nitroanilide (SAAAPN) and N-methoxysuccinyl-(ahmyl)~_pro-val-p-nitroanilide (MeSAAPVPN) in vitro and to determine their effects on an emphysema model in rive.
Materials and Methods FILE was purchased from Elastin Products Inc. (Pacific, M e ) ~zt-antitrypsin inhibitor, c h o n d r o i t i n 'qllfate, and lipids were from Sigma Chemical Co. (MO), 13-cis retinoic acid was a gift f r o m I ltffl'mann-t,a Roche (Nutty, N J), In kinetic experimcnls with 2 synthetic nilroaniline peptide substrates ( S A A A P N ,:.md t'Vle,';,,kAIWPN. Sigma Chemical Co.. Me)). the reaction rate was determined in a U v i k o n ~,pectrophotometer using a modification o f the method o f Bieth el al. 14t in which the rate o f p-nitroaniline l\)rmation is measured. To a disposable cuvette containing 0.9 ml o f 0.01 M lris-llCt. 0.15 M NaCI, pH g.0. 0, I ml lipids or liposome solulions in buffer were added to the desired linal concentration and 0.06 /zM enzyme added and preincubated for 5 rain tit 37
