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K G Wagner

Publications and source records attributed to K G Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1981

The ATP substrate site of a cyclic-nucleotide-independent protein kinase from porcine liver nuclei.

The ATP substrate site of a second messenger-independent protein kinase of the type NII from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP; they range from 4 microM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP-dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into delta delta values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions. With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its beta-phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N-6 amino group is required in the case of the cAMP-dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N-6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.

Adenosine Triphosphate↗
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↗
PubMed1980

Purification and properties of a DNase inhibitor from Nicotiana tabacum cell cultures.

Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE-cellulose and gel filtration resulted in a homogeneous protein (Mr = 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1:1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor X DNase-I complex have been calculated. This interaction is strongly temperature-dependent; at 20 degrees C and 26 degrees C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.

Cells, Cultured↗
PubMed1978

Mechanism of activation of protein kinase I from rabbit skeletal muscle. The equilibrium parameters of ligand interaction and protein dissociation.

Protein kinase I from rabbit skeletal muscle binds adenosine 3':5'-monophosphate (cAMP) and ATP with high affinity; cAMP promotes and ATP retards dissociation (activation) of the tetrameric enzyme. The interrelationship of ligand interaction with protein dissociation has been probed by quantitative ligand binding, using the filter assay technique, and by computer simulation of binding curves in terms of Scatchard plots. A comparison of the experimental and computed binding data strongly confirms the supposition that the dimeric regulatory subunit (R2) binds cAMP cooperatively. Mainly from ATP binding it is further concluded that the interaction of the two catalytic subunits with R2 is also strongly cooperative, whereas the binding of ATP to the holoenzyme is non-cooperative. The underlying random model, possessing low-affinity and high-affinity sites not only for ATP but also for cAMP, allows the determination of a minimal set of equilibrium parameters required for description and also an estimation of their magnitude. It further provides a basis for an explanation of the different behaviour of protein kinases I and II reported in the literature.

Animals↗
PubMed1978

Mechanism of activation of protein kinase I from rabbit skeletal muscle. Mapping of the cAMP site by spin-labeled cyclic nucleotides.

Binding of adenosine 3':5'-monophosphate (cAMP) to protein kinase (type I) from rabbit skeletal muscle has been investigated using spin-labeled cAMP derivatives. Different compounds were synthesized with the spin label attached by spacer chains of different length at different positions on the adenine base. Immobilization of the spin label, determined by comparing the electron-spin resonance spectra recorded in the presence of the kinase with those of the free ligand in solutions of different viscosities, gave information about the geometry of the cAMP site. Strong immobilization of the N-6 substituents up to a spacer length of seven atoms indicates a rather deep cleft of the cAMP site. The depth of this cleft differs, however, when the spin label is attached to the different positions at the adenine (N-6, C-2 and C-8). Whereas the N-6 derivatives indicate a rather deep site, the C-2 derivatives reveal a significantly smaller depth and C-8 substituents (syn conformation) obviously occupy a very shallow surface with almost no immobilation. In addition the binding affinities of the spin-labeled cAMP derivatives have been determined, together with those of a series of (diamagnetic) C-2 derivatives bearing hydrophobic alkyl chains of different length. The latter results helped to clarify the differences between the regions near to C-2 and N-6, respectively, of the cAMP site. N-6 spin-labeled derivatives have also been investigated in the presence of ATP and protein kinase. These results are interpreted as indicative of a conformational change at the cAMP site upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.

Animals↗
PubMed1977

Mechanism of activation of the protein kinase I from rabbit skeletal muscle. The high-affinity ATP site of the holoenzyme.

The high-affinity binding site for ATP of the holoenzyme of cAMP-dependent protein kinase (type I) from rabbit skeletal muscle has been investigated. Binding affinity of a series of ATP derivatives substituted at different sites in the molecule was determined by competition with tritiated ATP. The results were compared with data available from cAMP derivatives with the same substituents, in order to analyse the electronic and steric features of these two sites on the protein kinase. The comparison revealed significant differences of the effect of substituents towards the two sites. In particular the N6-derivatives of ATP and substituents affecting the gamma-phosphate indicate that the high-affinity ATP site of the protein kinase has similar properties as those found for phosphotransferase sites. The present results are consistent with the supposition that the high-affinity site for ATP on the holoenzyme is congruent with the phosphotransferase site of the catalytic subunit. Upon combination of catalytic and regulatory subunits this site would be transformed into a high-affinity site for ATP with simultaneous blocking of the phosphotransferase activity.

Adenosine Triphosphate↗
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

Specificity of DNA basic polypeptide interactions. II+ Influence of aromatic amino acid residues investigated with agarose bound lysine copolypeptides.

Binding affinities towards DNA and base pair specificities of lysine copolymers, containing different amounts of Phe, Tyr or Trp residues, were estimated using a previously described chromatographic method. Incorporation of few aromatic residues into polylysine causes a decrease in the binding affinity, however, further raising the aromatic residue - lysine ratio results in a continous increase of affinity, which is most pronounced with the Tyr copolymers and not observed with polymers containing neutral aliphatic amino acid residues. AT-specificity increases concomitant with binding affinity in the case of the Tyr copolymers but not with the Phe copolymers. The interaction of DNA with the alternating Phe-Lys polymer is significantly stronger than with the random copolymer of equal residue composition. The molecular and conformational reasons determining specificity are discussed.

Base Sequence↗
PubMed1976

Cooperative binding of fluorescein-labeled clupeine by DNA.

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.

Binding Sites↗
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↗