[Frontiers in Bioscience 3, a23-26, May 1, 1998]

	[Frontiers in Bioscience 3, a23-26, May 1, 1998] Reprints PubMed CAVEAT LECTOR
	AMINOALKYLAZIRIDINES AS SUBSTRATES AND INHIBITORS OF LYSYL OXIDASE: SPECIFIC INACTIVATION OF THE ENZYME BY N-(5-AMINOPENTYL)AZIRIDINE Narasimhan Nagan¹, Patrick S. Callery² and Herbert M. Kagan¹ 1 Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, and the ² Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506 Received 4/2/98 Accepted4/6/98 3. MATERIALS AND METHODS 3.1 Enzyme purification and assay Lysyl oxidase was purified to apparent homogeneity from calf aortas as previously described (16). The product migrated as a single band equivalent to a molecular mass of 32,000 Da when analyzed by sodium dodecyl sulfate gel electrophoresis (17). The purified enzyme preparation consists of four, 32 kDa ionic variants exhibiting a high degree of structural similarity, essentially the same substrate specificities and which appear to operate by the same catalytic mechanism (18,19). Aminoalkylaziridines were synthesized and purified as described (20,21). The specific activity of lysyl oxidase was determined by a tritium release assay against 125,000 cpm of a chick aortic elastin substrate labeled in organ culture with L-(4, 5-³H)lysine (22). The specific activities of purified enzyme preparations ranged from 400,000 to 800,000 cpm (³H)H₂O released per mg^-1 protein per 2 h of assay. All activities were corrected for background rates of tritium release of enzyme-free controls, and all activities were fully inhibited by 50 mM b-aminopropionitrile. Enzyme activities were also determined using a peroxidase-coupled fluorometric assay to obtain initial rates of lysyl oxidase-catalyzed H₂O₂ formation using 2.5 mM 1,5-diaminopentane, or other alkylamines, as specified, in assay mixtures containing 0.25 mg of homovanillic acid, 40 mg of horseradish peroxidase, and 1.2 M urea (23). Assays were initiated by the addition of lysyl oxidase to otherwise complete assay mixtures pre-equilibrated at the optimal assay temperature of 55^oC.

[Frontiers in Bioscience 3, a23-26, May 1, 1998]
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AMINOALKYLAZIRIDINES AS SUBSTRATES AND INHIBITORS OF LYSYL OXIDASE: SPECIFIC INACTIVATION OF THE ENZYME BY N-(5-AMINOPENTYL)AZIRIDINE

Narasimhan Nagan¹, Patrick S. Callery² and Herbert M. Kagan¹

1 Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, and the ² Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506

Received 4/2/98 Accepted4/6/98

3. MATERIALS AND METHODS

3.1 Enzyme purification and assay

Lysyl oxidase was purified to apparent homogeneity from calf aortas as previously described (16). The product migrated as a single band equivalent to a molecular mass of 32,000 Da when analyzed by sodium dodecyl sulfate gel electrophoresis (17). The purified enzyme preparation consists of four, 32 kDa ionic variants exhibiting a high degree of structural similarity, essentially the same substrate specificities and which appear to operate by the same catalytic mechanism (18,19). Aminoalkylaziridines were synthesized and purified as described (20,21).

The specific activity of lysyl oxidase was determined by a tritium release assay against 125,000 cpm of a chick aortic elastin substrate labeled in organ culture with L-(4, 5-³H)lysine (22). The specific activities of purified enzyme preparations ranged from 400,000 to 800,000 cpm (³H)H₂O released per mg^-1 protein per 2 h of assay. All activities were corrected for background rates of tritium release of enzyme-free controls, and all activities were fully inhibited by 50 mM b-aminopropionitrile. Enzyme activities were also determined using a peroxidase-coupled fluorometric assay to obtain initial rates of lysyl oxidase-catalyzed H₂O₂ formation using 2.5 mM 1,5-diaminopentane, or other alkylamines, as specified, in assay mixtures containing 0.25 mg of homovanillic acid, 40 mg of horseradish peroxidase, and 1.2 M urea (23). Assays were initiated by the addition of lysyl oxidase to otherwise complete assay mixtures pre-equilibrated at the optimal assay temperature of 55^oC.