A novel pyruvate kinase (PK-S) from boar spermatozoa is localized at the fibrous sheath and the acrosome.
Boar spermatozoa contain a novel pyruvate kinase (PK-S) that is tightly bound at the acrosome of the sperm head and at the fibrous sheath in the principal piece of the flagellum, while the midpiece contains a soluble pyruvate kinase (PK). PK-S could not be solubilized by detergents, but by trypsin with no loss of activity. Purified PK-S as well as PK-S still bound to cell structures and soluble sperm PK have all kinetics similar to those of rabbit muscle PK-M1. The PK-S subunit had a relative molecular mass of 64 +/- 1 x 10(3) (n = 3), i.e. slightly higher than that of PK-M1, and carried an N-terminal extension (NH(2)-TSEAM-COOH) that is lacking in native PK-M1. Evidence is provided that PK-S is encoded by the PKM gene. Antibodies produced against the N-terminus of purified PK-S (NH(2)-TSEAMPKAHMDAG-COOH) were specific for PK-S as they did not react with somatic PKs or soluble sperm PK, while anti-PK-M1 recognized both sperm PKs. Immunofluorescence microscopy showed anti-PK-S to label the acrosome and the flagellar principal piece, whereas the midpiece containing the mitochondria was labelled only by anti-PK-M1. Immunogold labelling confirmed the localization of PK-S at the acrosome. In the principal piece, both polyclonal anti-PK-M1 and anti-PK-S were found at the fibrous sheath. Our results suggest that PK-S is a major component in the structural organization of glycolysis in boar spermatozoa.
Feiden
Sandra
S
Molecular Physiology Section, Institute of Zoology, Johannes Gutenberg-University, Becherweg 9-11, D-55099 Mainz, Germany.
Stypa
Heike
H
Wolfrum
Uwe
U
Wegener
Gerhard
G
Kamp
Günter
G
D000177:Acrosome / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000494:pharmacology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D011770:Pyruvate Kinase / Q000032:analysis* / Q000235:genetics; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D032961:Sperm Midpiece / Q000201:enzymology; D013082:Sperm Tail / Q000201:enzymology; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011770:Pyruvate Kinase