Mechanism of spectrin degradation induced by phenylhydrazine in intact human erythrocytes.
The exposure of human erythrocytes to phenylhydrazine results in the degradation of both monomers of spectrin, a major cytoskeleton membrane protein. The degradative process, characterized by a loss of spectrin without the appearance of high-molecular-weight products, either under reducing conditions or not, is almost complete in 10 min when a 5% erythrocyte suspension is treated with 1 mM phenylhydrazine. Under these conditions, we found a loss of 62.3 and 48.5% for the alpha and beta monomer, respectively. A similar degradative extent was obtained when the membrane ghost plus cellular free extracts, were dialyzed, and the membrane ghost plus hemoglobin was exposed to 1 mM phenylhydrazine for 10 min. The presence of different proteinase inhibitors and effectors, such as EDTA, diethylenetriaminepentaacetic acid, EGTA, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, pepstatin, Ca2+ and ATP plus Mg2+, in the membrane ghost plus cellular free extract system (undialyzed) did not affect the degree of the spectrin-degradative process induced by phenylhydrazine. In addition, a purified spectrin tetramer preparation exposed to 1 mM phenylhydrazine in the presence of hemoglobin was degraded to an extent comparable to that with intact cells. Our data suggest that the initial degradative step of spectrin induced by phenylhydrazine in intact erythrocytes may be ascribed more to a direct oxidative breakdown, probably involving main-chain cleavage and side-chain cleavage processes, than to an eventual proteolytic system.
Arduini
A
A
Istituto di Scienze Biochimiche, Facoltà di Medicina, Università degli Studi G. D'Annunzio, Chieti, Italy.
Storto
S
S
Belfiglio
M
M
Scurti
R
R
Mancinelli
G
G
Federici
G
G
D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects*; D005609:Free Radicals; D006801:Humans; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000633:toxicity*; D011480:Protease Inhibitors / Q000494:pharmacology; D013049:Spectrin / Q000378:metabolism*
D005609:Free Radicals; D010659:Phenylhydrazines; D011480:Protease Inhibitors; C030299:phenylhydrazine; D013049:Spectrin