Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses

doi:10.1016/S0167-4838(98)00270-2

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1430, Issue 2, 19 March 1999, Pages 179-190

Biochimica et Biophy...

https://doi.org/10.1016/S0167-4838(98)00270-2 Get rights and content

Abstract

Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme–substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor–ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200–2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0°C, in the absence of significant catalytic activity, demonstrated two classes of binding sites (K_d=9.3×10⁻⁹ M and 2.5×10⁻⁷ M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component.

Introduction

Elastin displays extreme hydrophobicity and extensive cross-linking [1], [2], [3], [4], [5]. Because of these properties, it is quite stable and persistent in tissues unless subjected to the catalytic activity of one or more of a group of proteinases, collectively termed elastases because of their ability to solubilize amorphous elastin [6].

Solubilization of elastin represents a complicated biologic process since the substrate is exceedingly insoluble and is also heterogeneous with respect to enzyme reaction sites. Such a system is far more complex than the reaction of a proteinase with a soluble protein, and classical enzyme kinetics cannot be applied directly to the understanding of elastolytic mechanisms. However, because elastin turnover is important to human biology and disease [7], [8], [9], [10], studies of catalytic solubilization of elastin are of considerable interest. Studies of the binding of elastase to elastin assume particular biologic importance because it has been demonstrated that: (1) human leukocyte elastase (HLE) is bound to elastin in human emphysematous lungs [11]; (2) catalysis by HLE near sites of azurophil granule release in vivo likely occurs in quantum bursts with durations of tens of milliseconds, during which time substrate binding occurs [12]; and (3) HLE is relatively resistant to inhibition when bound to elastin [13], [14], [15], [16], [17], [18].

We have investigated various aspects of binding and catalytic activity of HLE on amorphous elastin. Using a model analogous to that widely applied to study ligand–receptor interactions, we have demonstrated the presence of two classes of HLE binding sites on elastin. Interaction of HLE with the higher affinity sites (K_d=9.3×10⁻⁹ M), which comprise only 6% of the total HLE binding capacity, leads to catalysis; moreover, this binding was very persistent. Binding to more prevalent, lower affinity sites on elastin was non-productive and rapidly dissociable. Furthermore, our results allow for comparisons and contrasts between the elastolytic activity of HLE and activity of vertebrate collagenase on other insoluble substrates (fibrillar collagens).

Section snippets

Materials and methods

Bovine ligamentum nuchae elastin (100–400 mesh, prepared as described by Starcher and Galione [19]) was obtained from Elastin Products, Pacific, MO. The elastin was tritiated using the method of Banda and colleagues [20] to specific activity 907 dpm μg⁻¹ elastin. Preliminary experiments indicated that the elastin was uniformly labeled, since the release of soluble ³H closely paralleled the release of soluble desmosine when the latter was measured by ion-pair HPLC [21].

Brij 35, deuterium oxide (D

Factors governing solubilization of ³H-elastin by HLE

Fig. 1A shows results obtained when ³H-elastin (320 μg) was incubated with increasing volumes of a constant concentration of HLE (0.2 μg ml⁻¹), such that the total amount of HLE in the reaction mixture increased 7-fold, while the enzyme concentration was unchanged. Substrate solubilization increased 3-fold despite the constant concentration of enzyme and a concomitant reduction in substrate concentration. Furthermore, the reaction rate was independent of whether the substrate was maintained

Discussion

The insoluble nature of elastin does not permit classical kinetic assessment of its interaction with HLE, since the analyses used in such studies relate only to enzymes and substrates in solution. In particular, the substrate concentration for an insoluble, heterogeneous substrate cannot be defined in classical terms. Although the kinetics of HLE interaction with a variety of soluble substrates have been assessed by classical means [29], [30], the substrate of greatest biologic relevance for

Acknowledgements

This research was supported by a grant from the Medical Research Council of Great Britain; by USPHS Grants HL29594, HL46440, and AM35805; by the Council For Tobacco Research, USA, Grants 1252, 1724, and 2843; by the British Lung Foundation; by the American Lung Association; and by Francis Families Foundation. C.A.O. is a Parker B. Francis Fellow in Pulmonary Research. The authors would like to thank Dr. John J. Jeffrey for helpful discussions.

References (53)

M. Wewers
Chest
(1989)
C.F. Reilly et al.
Biochim. Biophys. Acta
(1980)
B.C. Starcher et al.
Anal. Biochem.
(1976)
Y. Yamaguchi et al.
J. Chromatogr.
(1987)
D.Y. Twumasi et al.
J. Biol. Chem.
(1977)
J.C. Powers et al.
Methods Enzymol.
(1977)
H.G. Welgus et al.
J. Biol. Chem.
(1981)
R.P. Mecham et al.
J. Biol. Chem.
(1997)
R.P. Mecham et al.
Biochim. Biophys. Acta
(1979)
P.J. Stone et al.
Methods Enzymol.
(1982)

A. Baici

Biochim. Biophys. Acta

(1990)

G. Murphy et al.

J. Biol. Chem.

(1992)

H.G. Welgus et al.

J. Biol. Chem.

(1980)

K. Nakajima et al.

J. Biol. Chem.

(1979)

B.C. Starcher

Thorax

(1986)

L.B. Sandberg et al.

New Engl. J. Med.

(1981)

P.M. Gallop et al.

Physiol. Rev.

(1975)

D.R. Eyre et al.

Annu. Rev. Biochem.

(1984)

J. Rosenbloom

Lab. Invest.

(1984)

J.G. Bieth

Adv. Exp. Med. Biol.

(1984)

A. Janoff

Am. Rev. Respir. Dis.

(1985)

D.E. Niewoehner

J. Lab. Clin. Med.

(1988)

J.G. Bieth, in: R.P. Mecham (Ed.), Biology of Extracellular Matrix: Regulation of Matrix Accumulation, Academic Press,...

V.V. Damiano et al.

J. Clin. Invest.

(1986)

T.G. Liou et al.

Biochemistry

(1995)

W. Hornebeck et al.

Hoppe-Seyler’s Z. Physiol. Chem.

(1982)

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¹: Present address: Selly Oak Hospital, Raddlebarn Road, Birmingham, B29 6JD, UK.

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Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses

Abstract

Introduction

Section snippets

Materials and methods

Factors governing solubilization of 3H-elastin by HLE

Discussion

Acknowledgements

Chest

Biochim. Biophys. Acta

Anal. Biochem.

J. Chromatogr.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Methods Enzymol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Thorax

New Engl. J. Med.

Physiol. Rev.

Annu. Rev. Biochem.

Lab. Invest.

Adv. Exp. Med. Biol.

Am. Rev. Respir. Dis.

J. Lab. Clin. Med.

J. Clin. Invest.

Biochemistry

Hoppe-Seyler’s Z. Physiol. Chem.

Factors governing solubilization of ³H-elastin by HLE