Three-dimensional structure of herring sperm protamine Y-I with the aid of dark field electron microscopy. (1/10)

High resolution electron micrographs of herring sperm protamine (clupeine) Y-I provide sufficient detail to constrain the folding of the known amino-acid sequence of the protein into a unique three-dimensional configuration. The structure consists of a loose helix of turns of various sizes held together at one edge, spread apart along the other. This form appears to represent the shape of several fish protamines.  (+info)

Chemical synthesis of partially and fully phosphorylated protamines. (2/10)

Chromatographically purified components Z and YI of clupeine from herring have been phosphorylated by a chemical method. To enhance solubility in trimethylphosphate, the protamine capronate salts were used and phosphorylation was performed with POCl3. Both serine and threonine residues were phosphorylated; however, besides monophosphate esters considerable amounts of polyphosphate esters were obtained. Its nature was identified by 31P nuclear magnetic resonance spectroscopy and by chemical methods, such as comparison of the hydrolysis rate with that of ADP and phoshocreatine, respectively. The pyrophosphate bonds were cleaved by controlled hydrolysis with HCl. The phosphorylated protamine species were purified by gel filtration and chromatography on a Sephadex CM column. Fully phosphorylated clupeine Z (3 serine residues and 3 phosphates) and YI (3 serine, 2 threonine residues and 5 phosphates) were obtained as well as partially phosphorylated fractions; in the case of clupeine Z the resolution into mono, doubly and triply (fully) phosphorylated fractions was excellent. The overall yield of phosphorylated clupeine was better than 50%.  (+info)

Cooperative binding of fluorescein-labeled clupeine by DNA. (3/10)

The alpha-amino group of clupeine fraction Z from herring sperm was coupled with fluorescein. Binding of the labeled protamine by DNA is accompanied by significant fluorescence quenching up to 80%. This allowed the convenient determination of the binding behavior of protamine and DNA. Binding was found to be strongly cooperative and not be significantly affected by the size of DNA. The ionic strength dependence in the range up to 0.3 M NaCl was rather small. Binding parameters were derived according to classical unique-site treatment and to a concept which includes vagrant multi-site binding.  (+info)

Purification, properties and cation activation of galactosyltransferase from lactating-rat mammary Golgi membranes. (4/10)

Galactosyltransferase was purified from Golgi membranes of lactating-rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties. The enzyme occurred in both monomeric (43-46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N-terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro-Pro-Pro-Pro sequence. Kinetic studies demonstrated a high-affinity Mn2+-binding site (1) essential for activity, and a low-affinity Mn2+-binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase. It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.  (+info)

13C Nuclear magnetic resonance of protamines. The three main components of clupeine. (5/10)

The three main components (called YI, YII and Z) of clupeine a protamine from herring have been isolated and characterized by 13C nuclear magnetic resonance. Resonance belonging to the many different amino acid carbon atoms are observable and assigned. The effect of urea on the clupeine fractions points to the molecules being essentially in extended form in 2H2O solutions. Unusual properties of clupeine fractions can be observed in the presence of phosphate. Two of the fractions, i.e. YI and Z, show that a partial folding occurs in some well-defined whereas fraction YII remains essentially unaltered.  (+info)

Phosphorylated protamines. I. Binding stoichiometry and thermal stability of complexes in DNA. (6/10)

To decipher on a molecular level the role of protamine phosphorylation in spermiogenesis, clupeine Z species containing one, two or three serine phosphates were prepared utilizing a recently developed chemical procedure. The melting of complexes with calf thymus DNA showed that thermal stability decreases with increasing degree of phosphorylation. The stoichiometry of the nucleoprotamine complexes was investigated analyzing the melting curves and using the fluorescamine assay recently described. Phosphorylation significantly reduces binding stoichiometry defined as DNA-nucleotides covered by a protamine molecule. Thus, phosphorylated protamines are more densely packed along DNA; the implications on processes occurring in spermiogenesis as i. e. histone replacement, are discussed. A general discussion on the variability in protein-DNA stoichiometry values obtained by different procedures is included.  (+info)

Deoxyribonucleoproteins of herring sperm nuclei. I. Chemical composition. (7/10)

The chemical composition of deoxyribonucleoproteins from herring sperm nuclei was analyzed and the results are summarized as follows: 1. Chemical analysis of nuclear proteins and nucleic acids revealed that arginine/P molar ratio in herring sperm nuclei is unity but the ratio of arginine residues in protamine to phosphorus in the total DNA is 0.86. 2. The deoxyribonucleoproteins were isolated and their composition showed that about 14% of the total DNA in herring sperm nuclei is free from protamine and is bound with nonprotamine proteins in the weight ratio of nonprotamine proteins to DNA of 0.25-0.30. The remaining 86% of the total DNA is combined mainly with protamine and a small amount of nonprotamine proteins; the weight ratios of protamine and nonprotamine proteins to DNA are 0.75 and 0.08, respectively. In the latter complex, the molar ratio of arginine residues in protamine to phosphorus in DNA is unity.  (+info)

Nuclear magnetic resonance of protamines. A 13C relaxation study of the three main fractions of clupeine. (8/10)

The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques. The dynamic behaviour, examined through the evaluation of the spin lattice relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone alpha-carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 +/- 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side-chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase. Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.  (+info)

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