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PubMed1986

The effect of extracellular matrix modifications on UDP-glucose dehydrogenase activity in cultured human skin fibroblasts.

The effect of modifications of the extracellular matrix on the biosynthesis of glycosaminoglycans was investigated in human skin fibroblast cultures by studying UDPGDH activity in order to evaluate: a), the histoenzymological and biochemical modifications induced by chondroitinase ABC treatment (new experimental conditions were developed in order to obtain minimum cell damage); b), the reversibility of these modifications; c), the effect of growing the cells in the presence of chondroitinsulfate; d), the specificity of the modifications induced. The results demonstrated that our experimental conditions specifically affected intracellular UDPGDH activity. Chondroitinase ABC treatment induced a reversible increase of UDP-glucuronic acid synthesis. On the contrary, the presence of chondroitinsulfate in the growth medium completely inhibited UDPGDH activity.

Carbohydrate Dehydrogenases↗
PubMed1986

Carboxylate groups in bovine somatotropin involved in growth promoting activity. Theoretical models based upon individual kinetic constants to interpret the activity decay after chemical modification.

Modification of approximately one fifth of the carboxylate groups in bovine somatotropin with a water soluble carbodiimide caused loss of growth promoting potency pointing to the existence of residues related to the hormonal activity among those belonging to a fast reacting set. A sigmoidal curve was obtained whether the inactivation process was referred to reaction time or degree of modification. Isoelectrofocusing of derivatives released the native hormone from responsibility for the biological potency exerted by preparations with 1.5-2.6 modified carboxylate groups. Examination of the individual reaction kinetics of the 11 fast reacting residues, in turn, excluded the possibility of the sigmoidal character of the inactivation curve being caused by a nonexponential disappearance of essential residues, as a possible consequence of the chemical modification of others. According to synthetic models, the experimental curve may be the consequence of the effect of cumulative modification of 2 or 3 out of a set of 3 to 8 relevant residues.

Carboxypeptidases↗
PubMedDate not supplied

[Influence of the modification of Phe-tRNA synthetase from Escherichia coli by lysine- and arginine-specific reagent on the ionic interactions of the enzyme with tRNA Phe].

The influence of modification of Phe-RSase from E. coli MRE-600 by lysine- and arginine-specific reagent 2,4-pentandione on the Phe-RSase.tRNAPhe interactions was investigated. It was shown that modification of Phe-RSase with 2,4-pentandion leads to a decrease of the aminoacylation rate without any influence on the value of Km for tRNAPhe in this reaction and only a slight increase of the value of Kdiss for Phe-RSase.tRNAPhe complex. The log Km (Km-1)--ionic strength dependence for native enzyme and log Kdiss (K-1diss) for native enzyme and two forms modified on arginine and lysine residues were investigated. Results were interpreted quantitatively by Debye--Huckel approximation for two spherical macroions and by Daune approximation assuming that the region of tRNA implicated in ionic interactions is locally a cylindrical polyelectrolyte. It was shown that there are 2-4 electrostatic contacts in Phe-RSase.tRNAPhe interactions in limits of both approximations; modification of arginine residues in Phe-RSase doesn't change the number of electrostatic contacts, modification of lysine residues leads to an increase in the number of contacts. It was assumed that there are lysine residues in Phe-RSase essential for the tRNAPhe recognition. The possibility of participation of negative amino acid residues in electrostatic interactions with tRNAPhe is not excluded.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues.

Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.

1-Carboxyglutamic Acid↗
PubMedDate not supplied

[Post-synthetic modification of proteins].

Process of postsynthetic modifications of proteins in norm are considered. Two basic groups of modification processes are singled out: 1) processes which promote appearance of derivatives for 20 basic amino acids, i.e. change in the primary structure; 2) processes which are not associated with appearance of new amino acids but are responsible mainly for the changes in the polypeptide chain conformation and size. Modification processes of amino acid variations by means of methylation, acetylation, acylation, phosphorylation, ADP-ribosylation, glycosylation, amidation, hydroxylation and metal addition are described as referred to the first group. Proteolysis reactions are characterized in detail. Their significance for formation of biologically active peptides is considered. A notion "one gene--one protein" is thought to be incompetent because formation of a number of proteins necessitates participation of tens and hundreds of genes, coding enzymes of postsynthetic modification.

Acetylation↗
PubMed1985

Chemical modification of pig kidney 3,4-dihydroxyphenylalanine decarboxylase with diethyl pyrocarbonate. Evidence for an essential histidyl residue.

Diethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme.

Animals↗
PubMed1983

Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue.

Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.

Amino Acids↗
PubMed1984

Antigenic structure of hepatitis B surface antigen: identification of the "d" subtype determinant by chemical modification and use of monoclonal antibodies.

Hepatitis B surface antigens (HBsAg) of both the adw and ayw subtypes were reductively methylated with formaldehyde in the presence of sodium cyanoborohydride. The effect on antigenicity was determined by radioimmunoassay with monoclonal antibodies specific for seven different antigenic determinants. The reaction was shown to eliminate specifically the "d" antigenic activity of HBsAg/adw and to have no effect on HBsAg/ayw. Moreover, the reaction had only a slight affect on HBsAg/adw at one of the "a" antigenic determinants. The sites of modification were determined and the extent of modification of each site was compared to the loss of "d" antigenic activity. These studies demonstrated that the loss of "d" activity was due to the modification of lysine 122 in HBsAg/adw, and that although the amino terminus and lysine residues 141 and 160 of both HBsAg/adw and HBsAg/ayw are reactive, their modification does not alter any measurable antigenic activity.

Amino Acid Sequence↗
PubMed1981

Use of specific lysine modifications to identify the site of reaction between cytochrome c and ferricyanide.

The site of the reaction between horse heart ferrocytochrome c and ferricyanide was investigated by measuring the reaction rate of cytochrome c derivatives specifically modified at single lysine residues to form trifluoroacetyl or trifluoromethylphenylcarbamyl amino groups. Cytochrome c derivatives singly modified at lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice had rate constants decreased from that of native cytochrome c by factors of 1.29, 2.03, 1.12, 1.35, 1.46, 1.29, and 1.19, respectively. Modification of a given lysine with the bulky trifluoromethylphenylcarbamyl group caused nearly the same decrease in reaction rate as modification with the trifluoroacetyl group, indicating that the effect was due to removal of an electrostatic interaction between the protonated lysine amino group and ferricyanide. Modification of lysines 22, 55, 99, and 100 at the right side, bottom, and back of cytochrome c had no effect on the reaction rate. These results indicate that the reaction site is located at the exposed edge of the heme and that the electrostatic interaction between ferricyanide and cytochrome c is dominated by the lysine amino groups surrounding the heme crevice, which include lysine 86, in addition to the ones listed above. We have used the specific lysine modification results to estimate the contribution of each lysine amino group to the electrostatic interaction and have developed a semiempirical relation for the total electrostatic interaction.

Animals↗
PubMed1983

Immunological detection of carcinogen-modified DNA fragments after in vivo modification of cellular and viral chromatin.

Antibodies specific for DNA modified by (+/-)-trans-7, 8-dihydrobenzo(a)pyrene-7,8-diol-9, 10-epoxide have been used to quantitate the relative modification level in fragments derived from pBR322 DNA from cellular DNA and in the coding and noncoding strands of simian virus 40 DNA. DNA fragments with a covalent molar modification level ranging from less than 1 to over 200 are resolved by agarose gel electrophoresis and transferred to diazobenzyloxymethyl cellulose paper. The paper is incubated with antibodies specific to carcinogen-modified DNA, and the location of the antibody is visualized by autoradiography after incubation with 125I-protein A. The binding of antibodies is directly proportional to the level of DNA modification. Using this technique, we find that linker DNA is about 2.5- to 3-fold more accessible to (+/-)-trans-7,8-dehydrobenzo(a)pyrene-7, 8-diol-9, 10-epoxide than nucleosomal core DNA and that under in vivo conditions the coding and noncoding strands of the simian virus 40 chromosome are equally accessible to trans-7,8-dihydrobenzo[a]pyrene-7,8-diol-9, 10-epoxide. The approach described allows assessment of the relative level of modification in any DNA sequence which can be subjected to gel electrophoresis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1983

Protection of opiate receptors in NG108-15 against modification by N-ethylmaleimide.

Two different -SH groups associated with the opiate receptors of the mouse neuroblastoma X rat glioma hybrid NG108-15 have been identified. Modification of these by N-ethylmaleimide (NEM) (presumed to be via alkylation) or by para-chloromercuribenzoic acid (presumed to be via formation of mercury adducts) decreases the binding of both opiate agonists and antagonists to these receptors. Agonist binding is more sensitive than antagonist binding to modification by NEM. Losses in antagonist binding are accounted for totally by decreases in the number of binding sites; there are no corresponding losses in antagonist affinity. Losses of antagonist binding exhibit a pseudo-first order rate constant; the modification of only one such group completely destroys the binding site. Both agonists and antagonists protect against modification of this group by NEM. Sodium and lithium, but not GTP, also protect this group, indicating that the action of these monovalent cations is directly on the receptor moiety. Losses in agonist binding stem not only from decreases in receptor number but also from selective losses in affinity. This -SH group appears to be different from the one at the binding site as sodium, GTP, and antagonist ligands do not protect against losses in agonist affinity. Agonist high affinity also is lost in a pseudo-first order fashion indicating that an alteration of only one -SH group per receptor complex is sufficient to produce this effect. The possible roles of two sulfhydryls in opiate receptor function are discussed.

Animals↗
PubMed1984

The organization and complete nucleotide sequence of the PstI restriction-modification system.

We have determined the nucleotide sequence of a 4.0-kilobase DNA fragment containing the genes of the PstI restriction-modification system. Two large open reading frames were identified within the sequence and were ascribed to the restriction enzyme and methylase by the analysis of a series of deletion mutants. The two genes are encoded on opposite DNA strands, and hence must be transcribed from separate promoters rather than as a polycistronic message. The sequence of the first 10 amino acids of the restriction endonuclease was determined by sequential Edman degradation of the purified protein, permitting the alignment of the polypeptide with the DNA sequence. The NH2 terminus of the modification enzyme was established by sequential Edman degradation of the protein synthesized in bacterial minicells with different radiolabeled amino acids. The initiation codons of the two genes are separated by 130 base pairs. The deduced amino acid sequences indicate that the restriction endonuclease contains 326 amino acids with a calculated Mr = 37,370; the modification enzyme is composed of 507 amino acids with a calculated Mr = 56,830. There is no significant homology between the two proteins at the level of the primary structure. Antibody raised against the purified restriction endonuclease did not immunoprecipitate the modification enzyme. The transcription initiation sites were mapped using mung bean nuclease. Both of the transcripts begin with adenosine. The initiation sites are separated by only 70 base pairs. This close proximity suggests that the promoters for the two divergent genes overlap. DNase I protection experiments show that Escherichia coli RNA polymerase has a higher affinity for the methylase promoter than for the restriction enzyme promoter.

Amino Acid Sequence↗
PubMed1983

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites.

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.

Escherichia coli↗
PubMed1983

[Effect of modifications of a series of amino acid radicals on the enzymatic activity of glucoamylase from Aspergillus awamori].

The effect of chemical modification of various amino acid residues on the enzymatic activity of glucoamylase from Asp. awamori was studied. Modification of the carboxyl groups by taurine in the presence of water-soluble carbodiimide results in complete inactivation of the enzyme. The inactivation process includes two steps, namely non-specific modification and modification of the active center carboxyls. The rate constants of inactivation at both steps were measured in the presence and absence of the substrate, i. e. maltose. It was shown that the enzyme is inactivated by N-bromosuccinimide. Based on the data on the protection of the enzyme active center by the substrates (maltooligosaccharides of various lengths), it was concluded that the essential tryptophane residue(s) is localized in the fourth subsite. Ethoxycarbonylation, nitration and acetylation of glucoamylase do not change the catalytic activity of the enzyme. The protein was shown to contain no SH-groups.

Amino Acids↗
PubMed1984

Estrogen-dependent modification of ribosomal proteins. Effects of estrogen withdrawal on the distribution of constitutive and hormonally regulated mRNAs.

Estrogen-dependent modification of ribosomal proteins during induction of egg-yolk protein synthesis in avian liver was examined in vivo and in cultured hepatocytes. Modification of two proteins of the 40S ribosomal subunit was detected in vivo, within 40 min of injection of hormone. One of the proteins was identified as S6 and the other, tentatively, as S3a. Estrogen treatment resulted in the appearance of multiple, phosphorylated forms of S6 and a shift in electrophoretic mobility of the other protein that was consistent with its dephosphorylation. The steady state achieved within 2 h of injection could be maintained for up to 2 weeks when the hormone was administered from silastic implants. Removal of the implants resulted in a return to the preinduction state within 20-40 min. Similar modifications were induced in hepatocytes maintained in defined medium, with 17 beta-estradiol as the only hormonal supplement. In order to check on the possibility that the modifications observed could selectively influence mRNA utilization, the cytoplasmic distributions of two abundant mRNAs were monitored during the first few hours following withdrawal. One of these was serum albumin mRNA, the levels of which are unaffected by estrogen. The other was very-low-density apolipoprotein II mRNA which specifies a major egg-yolk protein. The synthesis of this mRNA is absolutely dependent on estrogen and its half-life is also markedly affected by the hormone.

Animals↗
PubMed1984

[Affinity modification of creatine kinase from the rabbit skeletal muscle by gamma-amide of ATP--a nitrogen mustard derivative].

A study of creatine kinase modification by ATP gamma-(N-(2-chloroethyl)-N-methyl)amide was performed. The attachment 1,7-1,8 moles of analogue per mole of functional dimer results in full inactivation of the enzyme. The substrates, ATP and ADP, protect the enzyme both against inactivation and covalent binding of analogue. The affinity modification rate depends on the reagent and magnesium ion concentrations and pH of the reaction mixture. The dissociation constants (1,0 and 1,5 mM) for the enzyme-analogue complexes and the affinity modification maximal rate constants (2,1 X 10(-3) and 1,2 X 10(-3) c-1) in the absence and presence of Mg2+ ions were estimated. Some differences in the affinity modification rates were observed for the nonidentical M and M'-subunits of creatine kinase. The data obtained are indicative of a histidine residue alkylation by the ATP analogue. This histidine (pK 7,7) may function as a general acid-base catalyst in deprotonation of the guanidinium group of creatine as the latter is phosphorylated by ATP.

Adenosine Triphosphate↗
PubMed1984

In vitro alterations similar to posttranslational modification of lens proteins.

Changes similar to posttranslational modifications that are observed with osmotic cataract formation in vivo can be seen with homogenates of lenses. These modifications are the loss of a 31,000 molecular weight (MW) beta crystallin polypeptide, an increase in a 25,000 MW membrane polypeptide. These modifications are potentiated by calcium and occur more rapidly at 37 degrees C than at 4 degrees C. Inhibitors of pepsin or of serine proteases do not influence these modifications, although proteolysis may be responsible for the changes observed.

Animals↗
PubMedDate not supplied

[Our modifications of urethrocystography].

The modifications of urethrocystography developed by the authors aimed at reducing possible damages but securing a good diagnostic value of the method. They made it possible by applying only one instead of the originally applied six catheterizations. Previously used barium sulphate is now replaced by urographin which is less damaging to the mucous membrane of the urinary bladder. In addition, a string of metal balls, which causes injury to the urethra, is no longer used. In the first modification a catheter into which a stainless steel spiral is inserted is used. The second modification is even simpler, because it is based only on the use of different urographin concentrations. By these modifications possible iatrogenic uroinfections are significantly reduced, there is no injury of the urethra, and the time needed for the preparation and carrying out of the method is shortened to one third. In X-ray pictures contrast is not so intense as is the case with barium and metal; they are a little lighter but quite good for diagnostic purposes.

Contrast Media↗