Basic proteins from sperm

As regards the structural features of galline, a protamine obtained from fowl sperm, some recent results with both unfractionated galline and some fractionated components are described in Chap. VII. B. 6, and those of a basic protein from bull sperm heads in Chap. VII. B. 7. 

Isolation and Partial Characterization of a Basic Protein from Bull Sperm Heads... 

CoELiNGH, ]. P., Rozijn, T. H., Monfoort, C. H. Isolation and partial characterization of a basic protein from bovine sperm heads. Biochim. biophys. Acta (Amst.) 188, 353—356 (1969). 

Nuclei were first isolated by Miescher (1869) from pus cells recovered from discarded surgical bandages. The principle constituent—a phosphorus-rich material then called nuclein—was stained by methyl green. A few years later salmon sperm were shown to contain a phosphorus-rich acidic compound—the nucleic acid—and a basic protein protamine. Further work by Kossel, Levene,... 

Protamine sulphate (from herring sperm) [9007-31-2] (a]p -85.5 (satd H2O). A strongly basic protein (white powder) with pKa values of 7.4-8.0 used to ppte nucleic acids from crude protein extracts. It dissolved to the extent of 1.25% in H2O. It is freely soluble in hot H2O but separates as an oil on cooling. It has been purified by chromatography on an IRA-400 ion-exchange resin in the SO form and washed with dilute H2SO4. Eluates are freeze-dried under high vacuum below 20°. This method is used to convert proteamine and protamine hydrochloride to the sulphate. [UV Rasmussen Z physiol Chem 224 97 1934, Ando and Sawada J Biochem Tokyo 49 252 1961, Felix and Hashimoto Z physiol Chem 330 205 1963]... 

A mixture of six similar very basic proteins (rich in arginine) isolated from trout sperm. 

In 1868 Friederich Miescher isolated a substance from the nuclei of pus cells that he named nuclein. A similar substance was subsequently isolated from the heads of salmon sperm. Nuclein was later shown to be a mixture of a basic protein and a phosphorus-containing organic acid, now called nucleic acid. There are two forms of nucleic acids, DNA and RNA. 

In general, the remodeling of sperm chromatin that accompanies stage I decondensation involves the replacement of sperm-specific basic proteins with histones from the egg and results in the formation of nucleosomes. It now seems clear, at least in amphibian egg and Drosophila embryo extracts, that this remodeling is mediated by specific factors that participate in both assembly and disassembly processes. These changes in chromatin composition and structure can be analyzed by one- or two-dimensional polyacrylamide gel electrophoresis and micrococcal nuclease digestion. 

Fig. 2 Two-dimensional PAGE analysis of sperm-associated proteins on condensed chromatin and following chromatin decondensation in egg extract and purified nucleoplasmin. Sperm nuclei were incubated in either (A) buffer, (B) Xenopus egg HSS, or (C) purified egg nucleoplasmin for 10 min. The nuclei were then pelleted, washed, and the chromatin-associated proteins were extracted with acid and analyzed in the first dimension by Triton-acid-urea (TAU)-PACE (left to right) and in the second dimension by SDS-PAGE (top to bottom). The positions of the core histones are indicated as H2A, H2B. H3. and H4 and the sperm-specific basic proteins as X and Y. The arrowheads and arrow indicate the positions of the cleavage-stage linker histone. B4, and Xenopus HMG-2, respectively (Dimitrov et at., 1994). [Reproduced from Philpott and Leno (1992) Copyright Cell Press.]...

Nucleoproteins are difficult to extract from mammalian spermatozoa this is not surprising because the nucleus of mammalian spermatozoa is protected against mechanical damage by a special keratinous membrane. The nuclear protein, like keratin, is rich in sulfur, but it contains more arginine than regular keratin. Studies on disrupted bull sperm nuclei revealed that the arginine content of mammalian sperm is intermediate between that of protamine and that of histone. Whether protamines are in fact present in mammalian sperm is not known. Analysis of a protein hydrolysate obtained after extraction of bull sperm does, however, indicate that a basic protein, relatively rich in arginine, is present in mammalian sperm. 

Nucleohistones are widely distributed in the somatic cell nuclei of animals, while nucleoprotamines are limited to the mature sperm cell nuclei of certain families of fish. Protamines have been separated from sperm nuclei of more than 50 species of fish and are usually named after the family of fish, according to the proposal of Kossel [1896 (1, 2)]. The main protamines are summarized in Table II-l together with the genus and species names of the organisms from which they are derived. It may sometimes be necessary to re-examine the protamines listed in the table to confirm whether they are true protamines from completely mature sperm nuclei or whether they are from immature ones, which contain basic proteins of various other types, as described below. 

Basic proteins of the histone type, or of a type intermediate between histone and protamine, are usually found in various amounts in cell nuclei of immature fish testes, often together with a protamine. Similar basic proteins of the histone type, but not protamines, are reported to be present in mature sperm cell nuclei of several fish and marine animals such as codfish (Stedman and Stedman, 1951), halibut, shad, sea urchin (Arbacia lixula and Stronglocentrotus lividus) starfish (Astropecten auran-tiacus and Echinaster spositus) (Kossel and Staudt, 1926), octopus (Eledone cirrosa) and some shellfish (Palau and Subirana, 1967). The same is true for some land animals such as Drosophila [Das et aL, 1964 (1,2,3)], grasshopper (Bloch and Brack, 1964 Das et al. 1965), and cricket (Kaye and McMaster-Kaye, 1966). No basic proteins can be readily extracted from mammalian sperm nuclei by acids (Dallam and Thomas, 1953). Nevertheless, there appears to exist in mammalian semen a basic keratin-like protein containing cystine in combination with DNA in a molecular ratio... 

The biosynthetic development of protamine was first studied by analysis of amino-acid composition and terminal groups of proteins in the nuclei fraction from the testis of rainbowtrout (Salmo irideus) at different stages of spermatogenesis, as shown in Table X-2. These studies showed that in the immature stage DNA is bound to basic proteins of the histone type, which are gradually replaced as maturation proceeds by basic proteins of the protamine type, so that in the fully matured sperm heads DNA is bound only to protamine [Ando and Hashimoto, 1958 (1 2, 3) Felix 1958 Felix, I960]. 

Nakano, M., Tobita, T., Ando, T. A basic protein, galline, from fowl sperm nuclei (in Japanese). Presented at 41st General Meeting of Japan. Biochem. Soc. (Tokyo, Oct. 28, 1968). Abstr. in Seikagaku (J. Japan. Biochem. Soc.) 40, 555 (1968). 

The biochemical investigation of DNA began with Friedrich Mescher, who carried out the first systematic chemical studies of cell nuclei. In 1868 Mescher isolated a phosphorus-containing substance, which he called nuclein, from the nuclei of pus cells (leukocytes) obtained from discarded surgical bandages. He found nuclein to consist of an acidic portion, which we know today as DNA, and a basic portion, protein. Mescher later found a similar acidic substance in the heads of sperm cells from salmon. Although he partially purified nuclein and studied its properties, the covalent (primary) structure of DNA (as shown in Fig. 8-7) was not known with certainty until the late 1940s. 

Heparin antagonism. Heparin effects wear off so rapidly that an antagonist is seldom required except after extracorporeal perfusion for heart surgery. Protamine, a protein obtained from fish sperm, reverses the anticoagulant action of heparin, when antagonism is needed. It is as strongly basic as heparin is acidic, which explains its immediate action. Protamine sulphate, 1 mg by slow i.v. injection, neutralises about 100 units of heparin derived from mucosa (mucous) or 80 units of heparin from lung ... 

---