Summary ― The presence of spectrin and ankyrin-like proteins was investigated during the differentiation and maturation of spermatozoa in mammalian species which have previously been studied for actin and calmodulin. These actin-binding proteins were characterized by immunoblotting and localized by immunoelectron microscopy. Neither spectrin nor ankyrin could be detected in the F-actin rich subacrosomal layer of spermatids in any species. In hamster and mouse maturing spermatids and spermatozoa, spectrin was mainly evidenced around the fibrous sheath of the flagellum whereas ankyrin was detected only in the neck. In rabbit spermatozoa, spectrin was evidenced in the outermost cytoplasmic layer of the post-acrosomal region and a light ankyrin labeling appeared in the neck. In rat, monkey and human sperm cells, these 2 proteins were not demonstrated. These results showed that as for actin there was no uniform pattern of distribution of spectrin and ankyrin among the 6 species studied.

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The active thiol group of lombricine kinase from Lurnbricus terrestris muscle was labelled with N-ethyl-[l-14C]maleimide. The resulting inactivated N-ethyl-[l-14C]succinimido enzyme was then subjected to tryptic hydrolysis.The peptide containing the labelled essential thiol group was isolated and found to contain:Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP : guanidine phosphotransferases previously studied, arginine kinase from Homarm vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. I n addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight N 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.Seven types of guanidine phosphotransferases with different guanidine substrate specificity are known [l]. All of them possess one or two sulfhydryl groups essential for their activity [2].The amino acid sequences around the two essential cysteinyl residues of creatine kinases from rabbit During a comparative study of the active site of these homologous enzymes, the primary structure around the thiol group of Homarus vulgaris muscle arginine kinase (mol. wt = 40000) was investigated in this laboratory [S].I n the present work we describe the isolation and the amino acid sequence determination of the tryptic peptide containing the essential cysteinyl residue obtained from a third guanidine phosphotransferase, lombricine kinase from Lurnbricm terrestris muscle (mol. wt = 80000).Among the other enzymes of this group, lombricine kinase is of peculiar interest ; further studies demonstrated that, although possessing a molecular weight of 80000 like creatine kinase . This pecularity suggests that only one of the two subunits of this enzyme plays a catalytic role. EXPERIMENTAL PROCEDURE MATERIALSPure lombrinic kinase from Lurnbricus terrestris was prepared as previously described [lo]. Its specific activity was 77.Trypsin(TPCK-trypsin) treated with tosyl phenylalanine chloromethyl ketone, obtained from Calbiochem, was dissolved in 1 mM HC1 at a concentration of 0.5O/, and stored a t -10 "C.