Diisopropyl fluorophosphate labeling of sperm-associated proteinases

Journal Article · Thu Feb 01 04:00:00 EST 1990 · Biology of Reproduction; (USA)

DOI:https://doi.org/10.1095/biolreprod42.2.329· OSTI ID:7016569

Odem, R R; Willand, J L; Polakoski, K L ^[1]

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1. Washington Univ. School of Medicine, St. Louis, MO (USA)

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

• OSTI ID: 7016569
• Journal Information: Biology of Reproduction; (USA), Journal Name: Biology of Reproduction; (USA) Vol. 42:2; ISSN BIREB; ISSN 0006-3363
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ANTIBODIES
AUTORADIOGRAPHY
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
CARBON ISOTOPES
ELECTROPHORESIS
ENZYME INHIBITORS
ENZYMES
FERTILIZATION
FUNCTIONS
GAMETES
GERM CELLS
HYDROLASES
ISOTOPES
KINETICS
LABELLING
MAMMALS
MAN
MOLECULAR STRUCTURE
PEPTIDE HYDROLASES
PRECURSOR
PRIMATES
REACTION KINETICS
SPERMATOZOA
VERTEBRATES