Primary structure of protamine from the Northern pike Esox lucius

Abstract

The basic nuclear protein in the sperm of the Northern pike is a protamine, 32-residues long, which behaved as a single component during ion-exchange chromatography and gel electrophoresis. Amino acid analysis gave close to molar ratios for the eight different residues with no evidence of microheterogeneity. However, the presence of sequence variants was revealed following a combination of automated protein sequencing and cleavage of the protamine by CNBr, endoproteinase Lys-C and thermolysin.At position 28 there is an equal probability of having serine or glycine. At position 9 glycine is found more frequently than serine. The reciprocal nature of the substitutions results in glycine and serine contents which are close to a 4:2 ratio. Pike protamines are homologous to those of the trout but show less sequence variation between components.There is considerable variation in the nature of basic proteins that bind DNA in sperm [l]. Bloch has classified these proteins into four groups, one of which comprises the arginine-rich protamines that lack cysteine [l]. This latter group is present in many fish such as the trout, herring, tuna fish [2] and dog-fish [3] but is absent from others like the carp [4] and winter flounder [S]. One of the characteristics of these protamines is that they are made up of several components which usually differ slightly in charge and length. The nature of this heterogeneity has been demonstrated in protamines from several species by protein sequencing [2,6] and confirmed for the trout protamines by DNA sequencing [6 -81. However, there is no obvious explanation for the existence of multiple components and no indication that they serve distinct functions in sperm chromatin.Our initial characterization of the protamine from the Northern pike Esox lucius did not reveal any obvious heterogeneity [9]. The protein behaved as a single component during gel electrophoresis and ion-exchange chromatography, and had an amino acid composition which could be reduced to a whole number ratio based on a length of 32 residues. In order to investigate further the question of heterogeneity in pike protamine we undertook to determine its amino acid sequence. MATERIALS AND METHODSNorthern pike were collected from West Lake, Prince Edward County, Ontario, in November. Testes were removed from freshly caught fish and frozen immediately on solid COZ. Salmon protamine was purchased from Sigma Chemical c o . Purification of protarninePreparation of testis nuclei and extraction of acid-soluble proteins were as described previously [5]. Pike protamine was purified from traces of other acid-soluble proteins by chromatography on carboxymethyl-cellulose (CM52 from Whatman). A column of bed volume 15 ml was equilibrated in 0.1 M LiC1/20 mM ammonium acetate (pH 5.0) and loaded with acid-soluble proteins (20 mg) in the same solution. After washing the column with 0.7 M LiC1/20mM ammonium acetate, protamine was eluted by increasing the LiCl concentration to 1.4 M. This latter fraction was dialysed in Spectrapor 3 membrane...

On the evolution of protamines in bony Fish: Alternatives to the ?Retroviral horizontal transmission? hypothesis

“…(1973); (4)Ando and Watanabe (1969); (5)Oliva and Dixon (1991); (6) Hoffman et al (1990); (7)Speckert et al (1983); (8)Bretzel (1972b); (9) Bretzel (1972a); (10) Bretzel (1973a); (l 1) BretzeI (1973b); (12)Okamoto et al (1992); (13)Okamoto et al (1987); (14)Chao and Davies (1992); and (15)Saperas et al (1993c).…”

The primary structure of a chondrichthyan protamine: A new apparent contradiction in protamine evolution

“…The association of arginine residues in clusters seems to confer a cooperative interaction between protamines and DNA (Willmitzer and Wagner 1980). Furthermore, when arginine clusters are established, a disruptive mutation (for instance RRRR --~ RRKR) is not readily accepted (Yulikova et al 1979;Speckert et al 1983). From this point of view, the presence of all three types of basic amino acid residues (R, K, H) in a given protamine, as well as the presence of heterogeneous clusters of basic amino acids (RKRK, KKKRK, etc.…”

Structure and expression of the highly repetitive histone H1‐related sperm chromatin proteins from winter flounder

“…Both mechanisms are used by fish. The majority of fish species, exemplified by the rainbow trout [2], northern pike [3] and yellow perch [4], replace their somatic histones with protamines. Others, like the grass carp [5], synthesize histone variants and maintain their sperm chromatin in a nucleosomal arrangement.…”