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Biomedical subjects

L Willmitzer

Publications and source records attributed to L Willmitzer.

132 records · Page 8Linked to original sources
PubMed1980

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.

Animals↗
PubMed1977

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.

Chemical Phenomena↗
PubMed1977

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.

Circular Dichroism↗
PubMed1977

On the competition between protamines and histones: studies directed towards the understanding of spermiogenesis.

Specific contacts between the crossinteracting histones effectively prevent their protamine-mediated displacement from nucleoproteins revealing a complex competition pattern not expected from their relative affinities to DNA. Histone H1 is the only species freed under a variety of conditions if native chromatin is used; the results found with chromatins obtained by conventional methods reflect the extent to which histone redistribution has occurred. It is concluded that the events occurring during spermiogenesis must be apt to disrupt histone interactions found in the nucleosomes. The possible role of a protamine phosphorylation/dephosphorylation mechanism is discussed on the basis of comparative experiments with protamines phosphorylated to different extents.

Animals↗
PubMed1975

Chemical synthesis of partially and fully phosphorylated protamines.

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%.

Amino Acids↗