Bioscience Reports 5, 473-476 (1985) 473 Printed in Great Britain Inhibition by ceruloplasmin of the cardiac sarcolemmal adrenochrome formation Carlo GUARNIERI and Carlo VENTURA Istituto Chimica Biologica, Centro Studi e Ricerche del Metabolismo Cardiaco, via Irnerio 48, 40126 Bologna, Italy (Received 23 April 1985) The addition of ceruloplasmin to bovine cardiac sarcolemmal vesicles supplemented with NADPH was able to reduce the formation of adrenochrome from adrenaline. This inhibitory effect appears at 2.5 IJM ceruloplasmin and it is almost complete at the level of 20 IJM. Previous research has demonstrated that the protein containing copper, ceruloplasmin, plays an important role as antioxidant in the extracellular space (1,2). The mechanisms proposed include the s activity of ceruloplasmin which, maintaining the iron in the Fe3+ state~ prevents the Fe 2+ stimulated lipid peroxidation process (3). In addition, ceruloplasmin can prevent copper ions from stimulating lipid peroxidation (4). Ceruloplasmin is also able to scavenge the superoxide radicals 02" by a noncatalytic reaction, less efficient than those produced by the enzyme superoxide dismutase (5). Among the various tissues~ the heart muscle seems to be particularly damaged by active oxygen metabolites generated in the extracellular space, for example by the activated neutrophils (6)7 or by the interaction of sarcolemma with catecholamines (7). Considering that the heart muscle contains receptors for cerulo- plasmin (g), the purpose of this study is to examine the possibility that ceruloplasmin may prevent the formation of adrenochrome induced in the cardiac sarcolemma by the superoxide radicals. Materials and Methods Ceruloplasmin (type VI human), catalase, superoxide dismutase and bovine albumin were from Sigma Chem. Corp. All other reagents were of the highest quality available form Merck. Bovine cardiac sarcolemma was prepared by the method of Reeves and Sutko (9), modified as suggested by Lamers and Stinis (10). After isolation, the vesicles were suspended in !60 mM KCI and 20 mM 3-(N-morpholine)-propane sulfonic acid, pH 7.t~ at a protein concentration of about 1.5-2 mg/ml and stored frozen in small aliquots at -S0~ The preparation was highly enriched in sarcolemmal membranes, as was revealed by the high specific activity of the marker enzymes ouabain sensitive Na+/K+-ATPase (65.8 t~mol/mg prot.h) and 5'-nucleotidase (28.9 tJmol/mg prot.h) (11). The formation of adrenochrome was monitored at 480 nm at 25~ in a