Denise Belaiche

Purification and characterization of nuclear basic proteins of human sperm

Highly purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins are in fact the same protein with different degrees of phosphorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.

Isolation and characterization of two protamines St1 and St2 from stallion spermatozoa, and amino-acid sequence of the major protamine St1.

Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.

Cuttlefish sperm protamines

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine-like components isolated from the sperm of bivalve molluscs.

Amino acid sequence of a cysteine-rich, arginine-rich sperm protamine of the dog-fish Scylliorhinus caniculus

Abstract Scylliorhinine Z2 is a small basic protein of 46 residues which has been purified from sperm nuclei of the dog-fish Scylliorhinus caniculus . It contains a high proportion of basic residues and of cysteine. When, compared to other protamines, it is characterized by the diversity of the amino acids represented in the molecule. The amino acid sequence shows that the N-terminal half is highly basic and that the hydrophobic residues are preferentially localized in the C-terminal region. The three residues with a hydrophilic side-chain are clustered and serine can occur in a phosphorylated forms; in mature sperm, the major fraction of the protamine is monophosphorylated, whereas mono- and diphosphorylated molecules are found during the terminal differentiation of the gamete within the testis. Scylliorhinine Z2 does not share common structural properties either with the other two scylliorhinines or with other fish protamines whose sequences have been previously determined.

Primary structure of the chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55

The DNA of the thermophilic archaebacterium Methanosarcina sp. CHTI 55 has been shown to be associated with two proteins called MC1 and MC2, of molecular mass 11 kDa and 17 kDa (Chartier et al. (1988) Biochim. Biophys. Acta 951, 149-156). The most abundant of these proteins, protein MC1, can protect DNA against thermal denaturation. In the present paper we report the covalent structure of protein MC1 and its effect on transcription of DNA in vitro. The covalent structure was determined from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid and arginine residues. The amino-acid sequence of protein MC1 from Methanosarcina sp. CHTI 55 is closely related to that of the protein MC1 (previously called HMb) isolated from Methanosarcina barkeri strain MS: among the nine substitutions observed between the two proteins seven are conservative. Transcription of DNA in vitro is stimulated by protein MC1 at low protein-to-DNA ratio but is inhibited at a ratio higher than 0.1 (w/w), which is the one determined in the bacterial deoxyribonucleoprotein complex.

Isolation and characterization of ATP-dependent proteolytically active ubiquitin in cock testis

1. We have successfully isolated and purified ubiquitin from cock testis by using an inhibitor, p-CMB (p-chloromercuribenzoate), which is one of the inhibitors specific for thiol-proteases and with the following procedures: heating up to 85 degrees C, ammonium sulfate fractionation, gel filtration on Sephadex G-75, chromatography on DE-52 and CM-11 and lyophilization. 2. Amino-acid analysis showed that Ub isolated from cock testis has 76 residues including 6 glycines. 3. Hydrazinolysis and carboxypeptidase digestion were also performed: the C-terminal residue is glycine. 4. The purity was checked by analytical SDS-PAGE and the isolated Ub exhibited only one band. 5. The Ub-dependent proteolysis experiment showed that this Ub was ATP-dependently proteolytically active. 6. In this paper we present evidence that a thiol enzyme is present during the purification procedure.

Cuttelfish Protamines: Amino-acid Sequences of Three Distinct Variants

Recent works (Wouters-Tyrou et al.,1988; Rousseaux-Prevost et al.,1988) have reported the existence of two nuclear protein transitions during the spermiogenesis of the cuttlefish Sepia officinalis. A transient protein (protein T) appears in round spermatids and is replaced by a typical protamine (protein Sp) in elongated spermatids.