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PubMed1990

[Effective selective modification of a single-stranded fragment of DNA with alkylating derivatives of short oligodeoxyribonucleotides in the presence of reaction effectors N-(2-hydroxyethyl)phenazine derivatives of oligodeoxyribonucleotides].

Tri-, tetra-, penta- and hexanucleotides bearing a reactive 4-(N-methylamino-N-2-chloroethyl)benzylamide group can effectively and selectively modify a single-stranded DNA fragment (302 nucleotides) in the presence of effectors, N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides complementary to DNA sequences adjacent to the binding site of the reagent. The reagents investigated modify not only single-stranded but also secondary-structured DNA regions. The modification extent depends on the length of oligonucleotide parts of the reagent and effector. A gap between the two stretches associated with the target DNA prevents the effector from functioning. The substitution of an octanucleotide effector by two tetranucleotide ones only slightly reduces the modification extent with a hexanucleotide reagent. A very efficient and specific modification can be achieved by using two effectors flanking the reactive oligonucleotide derivative. The approach leads to the modification extent of up to 89% with a hexanucleotide reagent.

Alkylating Agents↗

PubMed1990

Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase.

Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.

Adenosine Triphosphatases↗

PubMed1989

Stepwise modifications of keratin polypeptides during keratinization in palmar-plantar epidermis.

Precursor-product relationships among keratin polypeptides (cytokeratins) were studied in living and cornified layers of palmar-plantar epidermis. Serial 10 microns horizontal freeze-cut sections of punch biopsies were analysed by means of light microscopy and one-dimensional electrophoresis in a system that permitted identification of all major keratin polypeptides with apparent molecular weights over 60 kDa. Peptides that appeared to be related were compared by means of peptide mapping after partial proteolysis. The results suggest that both the basic-neutral (type II) cytokeratin no. 1 and the acidic (type I) cytokeratin no. 9 undergo two distinct modification steps with subsequent decreases in apparent molecular weight during keratinization. For both polypeptides the first modification appeared to take place and run to completion in close relation to the transition between the uppermost living epidermal layers and the lowest cornified layers. The second conversions of cytokeratins nos. 1 and 9 both appeared to take place within the stratum corneum but differed in two respects: 1) they appeared to start at different tissue sites; 2) whereas the second modification step appeared to comprise all cytokeratin 1 molecules, only a fraction of the cytokeratin 9 molecules passed through this step. These variations suggest that the different modification steps may be produced by different mechanisms that are regulated separately. It is concluded that the processing of cytokeratins during keratinization may be more complex than has previously been realized.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1989

Investigation of the relationship between tyrosyl residues and the adenosine 5'-monophosphate binding site of rabbit liver fructose-1,6-biphosphatase as studied by chemical modification and nuclear magnetic resonance spectroscopy.

The effects of AMP, fructose 6-phosphate (Fru-6-P), fructose 2,6-bisphosphate (Fru-2,6-P2), and paramagnetic ions on the aromatic region of the proton nuclear magnetic resonance (NMR) spectrum of rabbit liver fructose-1,6-bisphosphatase have been investigated at 300 MHz. Two well resolved peaks in this region of the NMR spectrum are assigned to the protons from the aromatic ring of a tyrosyl residue of the enzyme by chemical modification with tetranitromethane and by nuclear Overhauser effects. Nitration of the tyrosyl residue causes desensitization of the enzyme to AMP inhibition as well as the loss of activity. In the presence of AMP during the modifications, 1 tyrosyl residue could be protected, presumably the one observed by NMR. Binding of AMP, an allosteric inhibitor of the enzyme, to rabbit liver fructose-1,6-bisphosphatase leads to an upfield shift of the tyrosyl proton signals in the NMR spectrum. No chemical shift or line broadening could be detected in the presence of the paramagnetic manganous ion, Fru-2,6-P2, or Fru-6-P. The negative intramolecular nuclear Overhauser effect from the ribose H2' proton to the adenine H8 proton of AMP suggested that AMP binds to the enzyme with an anti conformation about the glycosidic bond. The failure to observe intermolecular nuclear Overhauser effects between the tyrosyl residue and the protons of AMP indicates that the distances between them are greater than 4 A. On the basis of these observations, it is suggested that the AMP-related tyrosyl residue may be close to the AMP binding site, but it is not directly involved in ligand binding. Rather, the protection of this tyrosyl residue by AMP as observed by chemical modification experiments may well be due to a conformational change that results from covalent modification of the enzyme.

Adenosine Monophosphate↗

PubMed1989

[Chemical modification of antibodies against carcino-embryonal antigen].

Chemical modification of different amino acid residues and the carbohydrate moiety of antibodies to carcino-embryonic antigen (CEA) was used to elucidate their role in the interaction with CEA and to evaluate the effect of antibody modification and steric factor in immunosorbent synthesis. The distribution of the modified groups among the structural fragments of IgG and the levels of changes in the antigen-binding properties of modified antibodies were investigated. The Fc-fragment of IgG was shown to contain carboxylic groups, tyrosine and histidine residues, whose modification influences the antigen-binding properties as a result of generalized conformational changes in the IgG molecule. A comparison of these results with earlier obtained data on the functional properties, of immobilized antibodies revealed that the decrease of antigen-binding characteristics of anti-CEA after IgG immobilization via NH2- and COOH-groups, carbohydrate moiety, tyrosine and histidine residues is due to the direct effect of antibody modification, whereas the changes in parameters of antibody interaction with antigen after IgG immobilization via SH-groups, methionine and histidine residues is due to steric hindrances.

Amino Acids↗

PubMed1989

[Modification of proteins by active oxygen and their degradation].

A detailed analysis of literary data concerning the oxidative modification of proteins by active oxygen species was carried out. It was shown that intermediate products of molecular oxygen reduction, e.g., superoxide anion radical, hydrogen peroxide and hydroxyl radical, can induce the inactivation of enzymes in vitro as a result of oxidative modification of certain amino acid residues necessary for the maintenance of native properties of the enzyme. In some cases modification of enzymes results in their degradation by proteolytic enzymes. Besides, some enzymes catalyzing the interconversions of active oxygen species (catalase superoxide dismutase, cytochrome P-450) are also inactivated in the course of catalysis under the oxidative action of active oxygen species. It was assumed that the oxidative modification of proteins appears to be one of the mechanisms which control their degradation in the cell. The hydroxyl radical oxidizing the amino acid residues located in the vicinity of the site of its synthesis is a direct modifying species. The superoxide anion radical and hydrogen peroxide are hydroxyl radical precursors and are responsible for the transport of oxidizing equivalents in the cell.

Animals↗

PubMed1988

Ligand-induced modification of a surface cAMP receptor of Dictyostelium discoideum does not require its occupancy.

In Dictyostelium discoideum amoebae, cAMP-induced phosphorylation of the surface cAMP receptor is associated with a discrete transition in its electrophoretic mobility. The native and modified forms of the receptor are designated R and D (Mr = 40,000 and 43,000). The relationship of the number of receptors which are modified as a function of the receptors which bind cAMP was investigated. Modification was assessed by determining the amounts of R and D forms in Western blots which detect all receptors whether or not they are exposed on the surface. Cyclic AMP or the analog, adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS), induced a loss of cAMP-binding activity (down-regulation), which was not accompanied by a loss of the receptor protein. About 60% of the receptors do not bind cAMP in the absence of Ca2+ and are unmasked by 10 mM Ca2+. However, the fraction of receptors which are modified in response to cAMP is equal in the absence or presence of Ca2+. (Rp)-cAMPs induces down-regulation (50%) but not modification. Addition of cAMP, following down-regulation by (Rp)-cAMPS, causes all receptors to be modified. cAMP induces both down-regulation (80%) and modification. Modification is more readily reversed than down-regulation: 30 min after removal of cAMP, receptors remain down-regulated (57%) but are found in the R form. All receptors shift to the D form when cAMP is readded to the cells. These results indicate that exposed, as well as cryptic and down-regulated receptors, are modified in response to the cAMP stimulus.

Calcium↗

PubMed1987

[Localization of the genes coding for the specific modification-restriction system in the E. coli tF strain].

The recombinant Escherichia coli tF strain has been shown to have its own specific modificational and restrictional system. In order to establish the location of genes, coding this system a plasmid has been isolated from the investigated strain, which substantiates the resistance to streptomycin. The plasmid DNA has been transformed into a recipient strain, E. coli O, which has no modificational and restrictional system of its own. The newly obtained E. coli O (ptF) transformants also have proved negative with regard to their testing for the presence of a specific modificational and restrictional system. The conclusion follows that the genes, coding the modificational and restrictional system of the E. coli tF strain are not located in the plasmid isolated from it.

Chromosomes, Bacterial↗

PubMed1988

Poliovirus-induced modification of host cell RNA polymerase IIO is prevented by cycloheximide and zinc.

Infection of HeLa cells with poliovirus results in a decrease in the level of RNA polymerase IIO, the transcriptionally active form of the enzyme, and a shutdown of host transcription (Rangel, L. M., Fernández-Tomas, C., Dahmus, M. E., and Gariglio, P. (1987) J. Virol. 61, 1002-1006). The effect of cycloheximide on poliovirus-induced modification of host RNA polymerase IIO was investigated. The inhibition of protein synthesis, at sequential stages during viral replication, prevents the modification of both total and chromatin-bound RNA polymerase IIO. Furthermore, the inclusion of zinc at a concentration that inhibits the proteolytic post-translational processing of viral polyprotein also prevents the modification of RNA polymerase IIO. These results suggest that host cell enzyme modification depends on the synthesis and processing of protein(s) encoded by the viral genome.

Cell Transformation, Viral↗

PubMed1987

Sites of covalent modification in Trg, a sensory transducer of Escherichia coli.

The Trg protein mediates chemotactic response of Escherichia coli to the attractants ribose and galactose. Like other transducers, Trg is a transmembrane protein that undergoes post-translational covalent modification. The modifications are hydrolysis (deamidation) of certain glutamine side chains to create glutamate residues and methylation of specific glutamates to form carboxyl methyl esters. Analysis of radiolabeled, tryptic peptides by high performance liquid chromatography and gas-phase sequencing allowed direct identification of the modified residues of Trg. The protein has 5 methyl-accepting residues. Four, at positions 304, 310, 311, and 318, are contained in a 23-residue tryptic peptide ending in lysine. The fifth, at position 500, is within a 25-residue tryptic peptide ending in arginine. At two sites, 311 and 318, glutamines are deamidated to create methyl-accepting glutamates. There is not a required order of modification among the sites. However, there is a substantial preference for methylation on the arginine peptide and, among sites on the lysine peptide, for the middle pair. Comparison of sequences surrounding modified residues identified in this work for Trg and previously for Tsr and Tar suggests a consensus sequence for methyl-accepting sites of Ala/Ser-Xaa-Xaa-Glu-Glu*-Xaa-Ala/OH-Ala-OH/Ala, where OH signifies Ser or Thr and the asterick marks the site of modification.

Amino Acid Sequence↗

PubMed1985

Modification of thiols of gizzard myosin alters ATPase activity, stability of myosin filaments, and the 6-10 S conformational transition.

The pattern of incorporation of [14C]N-ethylmaleimide (MalNEt) into gizzard myosin indicates the presence of two classes of thiols: rapidly and slowly modified. The first class contains two thiol residues, SH-A and SH-B, located in the myosin rod and the 17-kDa light chain, respectively, while the second contains at least two thiols located in the myosin heavy chain. Changes in ATPase activities upon modification occur rapidly or slowly, paralleling reaction of either the first or second class of thiols. Rapid changes include increases in the Ca2+- and Mg2+-activated activities of myosin alone, measured at ionic strengths below 0.3 M, and an increase and a decrease in the actin-activated activity of dephosphorylated and phosphorylated myosin, respectively. Modification of SH-A and SH-B with MalNEt is accompanied by stabilization of myosin filaments, seen as an increase in light-scattering intensity, and by destabilization of the folded, 10 S conformation of the myosin monomer. In the presence of 0.175 M NaCl and 1 mM MgATP, unmodified and MalNEt-modified myosin sediment in the ultracentrifuge as single components at 10.0 S and 6.0 S, respectively. The MalNEt-induced increase in the Ca2+- or Mg2+-activated ATPase activity, measured in the absence of actin, can be attributed either to stabilization of filaments or to destabilization of the 10 S conformation, depending on the ionic strength of the assay. Modification of the second class of thiols is accompanied by a decrease in K+-EDTA-activated activity and an increase in Ca2+-activated activity measured above 0.3 M NaCl, where myosin neither forms filaments nor assumes the 10 S conformation. These slow changes are characteristic of blocking the SH-1 thiols of skeletal-muscle myosin, but in gizzard myosin are attributable to modification of a less reactive thiol, SH-C.

Actins↗

PubMed1988

[Field-integrated dosage modification (FIDM). 2. The physical principles of the procedure].

A method is suggested and described which allows to introduce areas of different sizes, shapes, sites, and doses into a photon irradiation field. The bases for calculation and manufacturing of such irregular field stops containing integrated dose modification blocks are, according to precision requirements and data acquisition possibilities, either information provided by computed tomography or conventional planning radiographs. The following standard parameters are required as input data: sizes, shapes, and positions of the partial fields within the total irradiation field, depth of the reference plane, absorption coefficient of the modification material used, proportions of the planned dose modification, radiation quality, and other radiation field parameters. For the calculation of the dose distributions within the generally irregularly shaped irradiation volumes, an iterative algorithm has been formulated following the differential sector addition method and an equivalent TAR scheme. The quality of such a modifier can be checked by standard dosimetric methods. Deviations from the planned dose modification can be defined as sigma = +/- 5% for the radiation qualities employed (60Co gamma radiation and 15 MeV bremsstrahlung of a linear accelerator).

Gamma Rays↗

PubMed1988

Conformation-sensitive modification of the type II calmodulin-dependent protein kinase by phenylglyoxal.

Chemical modification by phenylglyoxal was used to investigate relationships between the structure, function, and regulation of the type II calmodulin-dependent protein kinase. Modification of the protein kinase by phenylglyoxal resulted in specific labeling of one distinct site, most likely an important arginine residue, with concomitant inactivation of the enzyme. Labeling and inactivation of the protein kinase was prevented by Mg2+-ADP which suggests that modification occurred at, or in close proximity to, its nucleotide-binding pocket. Half-maximal protection by Mg2+-ADP was enhanced by calmodulin which decreased the K0.5 for ADP from 540 to 61 microM. This response of the enzyme to calmodulin indicates that the modulator protein increases the affinity of the protein kinase for nucleotides. Inactivation of the enzyme by phenylglyoxal was dependent on the presence of Mg2+ or Ca2+/calmodulin, and further enhanced by the simultaneous addition of these effectors to the reaction. The Mg2+ effect is indicative of binding of this divalent metal ion to the protein kinase even in the absence of calmodulin and nucleotides. The stimulation of the modification reaction by calmodulin indicates an increase in the reactivity or accessibility of the modified residue in response to calmodulin-regulated conformational changes on the enzyme. The calmodulin-induced changes observed in this study may play important roles in the molecular mechanisms of activation of the type II calmodulin-dependent protein kinase.

Aldehydes↗

PubMed1988

[Crystal modifications of 3-cyan-6-methyl-5-(pyrid-4-yl)-1,2-dihydropyrid-2-one (milrinone)].

The preparation and characterisation of crystalline alpha-, beta- and gamma modifications of 3-cyan-6-methyl-5-(4-pyrid-4-yl)-1,2-dihydropyrid-2-on e are described. The thermic transformation of alpha- and beta-modification into gamma-modification was proved by thermogravimetric analysis (t.g.a.), differential scanning calorimetry (d.s.c.), IR and powder diffraction pattern. In dissolution behaviour no significant differences were found between the modifications.

Chemical Phenomena↗

PubMed1987

Chemical modification of histidine, tyrosine, tryptophan and cysteine residues in carp (Cyprinus carpio) muscle enolase.

Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.

Amino Acids↗

PubMed1987

Energy expenditure in the control of biochemical systems by covalent modification.

Regulation by reversible, covalent modification of proteins requires a continuous expenditure of energy, even in a steady-state situation. The cost of this energy drain is evaluated for the case of an effector controlling the modifying enzyme and an effector controlling the demodifying enzyme and for the case of dual control in which an effector activates one of these enzymes and inhibits the other. Energy consumption is determined when the converter enzymes are functioning in the first-order and zero-order domains. The profile of energy expenditure versus fractional protein modification at steady state varies both as a function of the mechanism of control of the converter enzymes and of the kinetic domain in which they operate. This theory allows one to predict the strategies that would minimize energy costs. Dual control appears to provide maximum sensitivity with minimal energy expenditure. The analysis is applied to two experimental systems. Comparison of ATP turnover rates with rates for individual modification enzymes in living systems shows that a significant fraction of the total energy expenditure of an organism is required for the large number of reactions which involve covalent modification of proteins. It is concluded that there will be selection pressure for energy-efficient control of covalent regulation.

Adenosine Triphosphate↗

PubMed1987

[The polymorphism of drugs in powders and tablets. 1. The preparation and characterization of polymorphic modifications of phenobarbital].

The characterisation of three phenobarbital modifications by thermic examination procedures (DSC, DTA) is being described. Modification I was obtained by thermic treatment of the brands (modification II) from Hungary and the GDR. The spray product prepared, consisting of very fine hollow spheres, was identified as modification III. Besides the particle size distribution the form of the particle was determined by scanning electron microscopy (REM). The best results regarding saturation solubility and speed of dissolution were found for the spray product.

Chemistry, Pharmaceutical↗

PubMed1987

Effects of chemical modifications on the surface- and protein-binding properties of the light chain of human high molecular weight kininogen.

The light chain of kallikrein-cleaved human high molecular weight kininogen is solely responsible for its cofactor activity in blood clotting. Sequencing of the NH2-terminal region of the light chain reported herein identified the third kallikrein cleavage site of high molecular weight kininogen as Arg-437. The co-factor activity of high molecular weight kininogen consists of the capacity to bind to negatively charged surfaces and to factor XI or prekallikrein. Chemical modification of the histidines by either photooxidation or ethoxyformic anhydride affected the equivalent of 14-16 of 23 histidines available and resulted in over 90% loss in procoagulant activity. The modified protein had drastically reduced surface- and zinc-binding capacity, but it bound successfully to either factor XI or prekallikrein. In contrast, modification of two carboxyl groups, which led to approximately 80-90% loss of procoagulant activity, seriously compromised protein binding but left surface binding unaffected. All 3 tryptophans were modified at pH 4.0 with N-bromosuccinimide with a 70% reduction in procoagulant activity, but only 1 tryptophan was available for reaction at pH 7.35, resulting in a 50% loss in activity. Tryptophan modification at acidic pH affected protein binding but did not modify surface or zinc binding. Modification of both available tyrosine and 9 of 18 available lysine residues did not have a significant effect on the procoagulant activity of the light chain. These studies indicate that histidines participate in surface binding and that free carboxyl groups and tryptophan side chains are involved in binding of high molecular weight kininogen to other clotting factors.

Amino Acid Sequence↗