N(omega)-phosphoarginine phosphatase (17 kDa) and alkaline phosphatase as protein arginine phosphatases.

Seven synthetic polymers, (Glu4, Tyr)n, (Arg)n, (Arg, Pro, Thr)n, (Arg-Gly-Glu)6, (Arg-Gly-Phe)6, (Glu-Arg-Gly-Phe)5, and (Ala-Leu-Arg-Arg-Ile-Arg-Gly-Glu-Arg)2, were treated with phosphoryl chloride to phosphorylate their Tyr, Thr, and Arg residues. Protamines and histones were phosphorylated similarly. These phosphorylated peptides were examined as to whether or not they serve as substrates for intestinal alkaline phosphatase [EC 3.1.3.1] and liver N(omega)-phosphoarginine phosphatase [Kuba, M., Ohmori, H., and Kumon, A. (1992) Eur. J. Biochem. 208, 747-752]. Phosphorylated polyarginine was hydrolyzed with a lower Km with alkaline phosphatase than with N(omega)-phosphoarginine phosphatase, while the phosphorylated forms of (Arg-Gly-Phe)6 and culpeine were better substrates for N(omega)-phosphoarginine phosphatase. When (Arg, Pro, Thr)n and culpeine were phosphorylated chemically after treatment with phenylglyoxal, these phosphorylated peptides were worse substrates for N(omega)-phosphoarginine phosphatase than for alkaline phosphatase. Moreover, the results of proton-decoupled 31P NMR analysis indicated that N(omega)-phosphoarginine phosphatase released Pi from N(omega)-phosphoarginine residues of phosphopeptides. These results indicate that both phosphatases function as protein arginine phosphatases in different manners, and that N(omega)-phosphoarginine phosphatase is useful for selectively detecting N(omega)-phosphoarginine residue in peptides containing various kinds of phosphorylated amino acids.

The use of affinity coelectrophoresis to characterize cooperative, nonspecific nucleic acid binding peptides.

Affinity coelectrophoresis (ACE) is a technique for characterizing ligand/nucleic acid binding interactions under equilibrium conditions. It is used here to characterize the protamine clupeine Z binding to several DNA fragments in order to define the use and limitations of ACE for ligands that bind cooperatively and nonspecifically to nucleic acids. The results demonstrate that the ACE data for cooperative, nonspecific ligands can be analyzed using the McGhee-von Hippel model and that binding-site sizes can be accurately determined using lattices containing as few as one site. However, binding constants can be greatly underestimated for some cooperative ligands with large-site sizes if small lattices are used. The salt dependence of the binding constant can also be determined but is limited to salt concentrations less than approximately 300 mM. Given the simplicity and reproducibility of the ACE assay, it should find many applications for studying binding interactions for a variety of cooperative and noncooperative nucleic acid binding peptides and proteins.

Structure and function of protamines: an 1H nuclear magnetic resonance investigation of the interaction of clupeines with mononucleotides.

Protamines form a class of low-molecular-weight proteins that protect the chromosomal DNA in the spermatic cells of eukaryotic organisms. Protamines are located in the small and/or large groove of DNA where they complex the DNA nucleotides. Very little is known up to date on the role and specificity of binding of the various protamine fractions belonging to a single eukaryotic species. In the present paper, a detailed investigation on the complexation properties of the protamine fractions (clupeines) extracted from herrings has been carried out by means of proton nuclear magnetic resonance and ultraviolet absorbtion data. In particular, the binding properties of the clupeine fractions with purinic (5'dAMP) and pyrimidinic (5'dCMP) mononucleotides have been measured and analysed at different clupeine concentrations. The results indicate that, contrary to previous preliminary hypothesis, the three clupeine fractions exhibit quite comparable binding properties toward mononucleotides. In addition it has been found that nucleotides can induce a conformational transition of the disorder-order type in the clupeine molecules and this property is concentration and temperature dependent. It is concluded that, as far as specificity is concerned, the clupeine fractions seem to possess the same behaviour toward mononucleotides.

Structure of porcine insulin cocrystallized with clupeine Z.

The crystal structure of NPH-insulin, pig insulin cocrystallized with zinc, m-cresol and protamine, has been solved by molecular replacement and refined using restrained least-squares refinement methods. The final crystallographic R factor for all reflections between 2 and 10 A is 19.4%. The insulin molecules are arranged as hexamers with two tetrahedrally coordinated Zn atoms in the central channel and one m-cresol bound to each monomer near His B5. One protamine binding site has been unequivocally identified near a dimer-dimer interface, although most of the polypeptide is crystallographically disordered. The conformation of the insulin moiety and the structural differences between the three unique monomers have been analysed. The zinc and m-cresol environments are described and the nature of the protamine binding site is outlined.

Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase.

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.

The binding mode of a mammalian (boar) protamine to DNA.

The binding modes of mammalian and fish protamines to DNA were studied by reconstitution experiments from dansylated protamines and DNA, using fluorescence spectroscopy, thermal denaturation and sedimentation. Both boar and fish protamines showed strong positive cooperativity in binding to DNA. Binding parameters of the protamines were determined in 0.1 M NaCl, 50 mM Tricine-HCl, pH 7.4, at 37 degrees C: in the boar protamine, the cooperative binding constant (Kc) = 3.4 X 10(6) M-1 and the cooperative factor (q) = 667, in the fish protamine, Kc = 1.8 X 10(7) M-1 and q = 304. The boar protamines bound to DNA with two functional domains, but the fish protamines bound directly to DNA as a single linear molecule.

The binding of protamines to DNA; role of protamine phosphorylation.

The thermodynamics of protamine-DNA interation was investigated with clupeine Z from herring labeled at its amino terminus with fluorescein. The ionic strength dependence, the influence of protamine phosphorylation, of the native DNA conformation, using native and heat-denatured DNA, and of the protamine primary structure, using two oligoarginine peptides of similar length as the clupeine, was thoroughly studied. The unusually high cooperativity of interaction found is strictly correlated to the native DNA conformation and the protamine primary structure. Cooperativity is explained by cross-linking of DNA segments resulting in an increase of the negative charge density. The importance of protamine phosphorylation lies in the fact that thermodynamically governed interaction with DNA and favorable cross-linking of DNA are shifted to physiologically reasonable ionic strengths.

Phosphorylated protamines. II. Circular dichroism of complexes with DNA, dependency on ionic strength.

The influence of protamine phosphorylation upon the conformation of nucleoprotamine complexes was studied at different ionic strengths using circular dichroism. The sharp onset of CD spectral changes upon decreasing the NaC1 concentrationwas correlated with the beginning of complex formation and can be used to determine apparent binding affinities in terms of a critical ionic strength. It is show that phosphorylation strongly reduces the binding strength of protamines towards DNA. Directly mixed and reconstituted complexes reveal differences in their CD spectra, which decrease with increasing ionic strength. Spectra of complexes between threefold phosphorylated clupeine Z and DNA obtained by reconstitution or direct mixing at higher ionic strength resemble the phi-type spectra of DNA and are unique for the phosphorylated species. The implications of protamine phosphorylation for chromatin or DNA condensation havebeen discussed.

Phosphorylated protamines. I. Binding stoichiometry and thermal stability of complexes in DNA.

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.

Preparation and enzymatic properties of subtilisin Novo chemically attached to soluble DEAE-dextran and insoluble DEAE-sephadex.

Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and Km values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifter towards the alkaline pH region and the Km values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40-65% reduction of kcat for hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester and benzyloxycarbonyl-glycyl-L-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40-60% of the subtilisin values. With Deae-sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can effect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes.