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PubMed1986

Intra- and extracellular modifications of apolipoproteins.

This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal peptide which is cleaved cotranslationally by the signal peptidase of the rough endoplasmic reticulum. ApoE is further modified intracellularly with carbohydrate chains containing sialic acid and is secreted in the modified form designated apoEs. The modified apoE is subsequently desialated in plasma. ApoA-I is secreted in a proapoA-I form, which consists of 249 amino acids. The N-terminal hexapeptide of proapoA-I is cleaved extracellularly by a proapoA-I to plasma apoA-I converting protease. This cleavage generates the plasma apoA-I form which consists of 243 amino acids. Other known apolipoprotein modifications include the modification of apoB, apoC-III, and apoD with carbohydrate chains that contain sialic acid and the proteolytic cleavage of the proapoA-II segment. At the present time we are able to distinguish several isoprotein forms for a particular apolipoprotein. In addition, we began to understand the biochemical changes which lead to a few of these isoproteins. Future research should be directed toward a better understanding not only of the structure but most importantly of the physiological significance of the different apolipoprotein forms.

Amino Acid Sequence↗
PubMed1992

Morbidity and recurrence with modifications of radical vulvectomy and groin dissection.

Vulvar carcinoma has been managed in recent years with modifications of radical vulvectomy and groin dissection. Separate groin incisions, superficial inguinal lymphadenectomy, unilateral groin dissection, and wide excision have been utilized to reduce the morbidity of treatment. In this study, the surgical management of 82 patients with vulvar squamous cell carcinoma was reviewed in order to assess morbidity and risk of recurrence. A modification of radical vulvectomy and groin dissection was employed in 67 patients, while 15 patients underwent classical en-bloc vulvar and groin dissection. Wound complications of the vulva occurred in 1 of 12 patients undergoing hemivulvectomy, in 8 of 55 undergoing radical vulvectomy, and in 7 of 15 who had en-bloc vulvar resection and groin dissection (P = 0.01). Among the 46 patients undergoing bilateral groin dissection through separate incisions, groin breakdown, lymphocyst, and lymphedema occurred in 10 (22%), 7 (15%), and 7 (15%), versus 0, 1 (7%), and 2 (13%) of the 15 who had unilateral groin dissection. Modification of vulvar resection did not increase the risk of local recurrence. Groin recurrence developed in 2 of 15 patients who underwent en-bloc groin dissection and in 1 of 46 who underwent bilateral groin dissection through separate incisions. Two of 15 who had a unilateral groin dissection recurred in the contralateral groin. The risk of recurrence as well as morbidity following modifications of radical vulvectomy with groin dissection should be considered when planning treatment.

Aged↗
PubMed1984

Two plasmid-determined restriction and modification systems in Streptococcus lactis.

Two restriction and modification systems were found in Streptococcus lactis strain IL594 which was found to contain 9 plasmids designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments, we showed that a restriction and modification system was related to the presence of pIL6 or pIL7. The pIL6-determined restriction and modification system was confirmed by cotransfer of the plasmid and of the restriction and modification system to a plasmid-free, nonrestricting, and nonmodifying derivative of S. lactis IL594.

Bacteriophages↗
PubMed1990

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation.

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.

HeLa Cells↗
PubMed1982

Specific chemical modification of the readily nitrated tyrosine of the RTEM beta-lactamase and of bacillus cereus beta-lactamase I. The role of the tyrosine in beta-lactamase catalysis.

The function of the hydroxyl group of the tyrosine residue readily nitrated by tetranitromethane (tyrosine-105) in the RTEM plasmid-derived beta-lactamase (penicillinase; penicillin amido beta-lactam-hydrolase, EC 3.5.1.6) from E. coli and in Bacillus cereus beta-lactamase I has been investigated by chemical modification methods. In the case of B. cereus beta-lactamase I the nitrated tyrosine can be acetylated by acetic anhydride without effect on beta-lactamase activity The nitrated tyrosine of the E. coli enzyme can also be acetylated but in this case beta-lactamase activity is lost in a manner which directly correlates with extent of acetylation. However, deacetylation of the nitrotyrosine does not restore activity. The dilemma created by the latter result has been resolved by development of a new method of tyrosine hydroxyl modification at low pH. The nitrated enzyme is reduced by dithionite and then treated with either carbonyldiimidazole or N-(2.2.2-trifluoroethoxycarbonyl)imidazole, both of which convert 3-aminotyrosine into benzoxazolinonylalanine. That the final modification has been achieved is demonstrated both by classical chemical methods and by employment of Fourier transform infrared spectroscopy to detect the characteristic benzoxazolinone carbonyl absorption. Further, it is shown that no significant loss of beta-lactamase activity is associated with this modification. Hence in neither the B. cereus or the E. coli enzyme does the readily nitrated tyrosine residue have a direct chemical function at the beta-lactamase active site.

Acetylation↗
PubMed1983

The modification with tetranitromethane of an essential tyrosine in the active site of pig fumarase.

Modification of pig heart fumarase (L-malate hydro-lyase, EC 4.2.1.2) with tetranitromethane results in loss of enzymatic activity. The inactivation is slowed down in the presence of substrates, indicating that the modification reaction takes place at the level of the substrate binding sites. From these inactivation kinetics, a value Kd = 78 microM is calculated for the mixture of substrates (L-malate + fumarate). This is in fairly good agreement with the Michaelis constant Km = 31 microM. Spectrophotometric data indicate that modification of one tyrosine residue per fumarase subunit is responsible for the inactivation; one or more additional residues, which do not participate in the binding sites, are modified at much lower rates. Amino acid analyses confirm the presence of nitrotyrosine and exclude the possibility of tetranitromethane-mediated polymerization side-reactions. It is concluded from the pH-dependence of the nitration reaction that the inactivation of fumarase is not caused by cysteine modification. Additional studies of nitration of melittin, a tryptophan-containing model peptide, are described. From the absorption spectra of modified melittin, in comparison with the spectra of nitrofumarase, it is concluded that the tryptophan residues of the latter enzyme remain intact during the reaction with tetranitromethane. Finally, evidence is given for an independent action of the four fumarase subunits, i.e., inactivation of one subunit does not influence the catalysis by the other three subunits. Moreover, it is shown that only fumarase tetramers with all four subunits nitrated are unable to bind to a Sepharose-pyromellitic acid affinity column.

Amino Acids↗
PubMed1984

Chemical modification of dopamine beta-hydroxylase.

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.

Amino Acids↗
PubMed1986

Specific, limited tryptic modification of wheat-germ fructose-bisphosphate aldolase subunits: destruction of catalytic activity but not of ability to establish precise subunit-subunit recognition.

We have been using the glycolytic enzyme fructose-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) as a model system to investigate the assembly of oligomeric enzymes. In the present work, we investigate the effect of specific, limited tryptic modification on the properties of aldolase isolated from wheat germ. The wheat-germ enzyme was selected, since several aldolases isolated from animal sources were not readily susceptible to the specific tryptic modification seen with this plant enzyme. We will show that: Low levels of trypsin cause a first-order inactivation of wheat-germ aldolase activity which is associated with a fairly specific cleavage of the enzyme which reduces its subunit molecular weight from 41000 to 39000. The proteolytic modification is greatly inhibited in the presence of the aldolase substrate, fructose bisphosphate. The intact and modified enzymes appear to have similar surface changes, as judged by their behavior during electrophoresis in polyacrylamide gels under non-denaturing conditions. The modified aldolase is not specifically eluted from phosphocellulose columns by fructose bisphosphate under the conditions used in the affinity chromatographic isolation of the intact enzyme, suggesting that the modified enzyme may no longer be able to bind substrate. Although enzymatically inactive, the modified aldolase subunits are able to refold and reassociate into tetrameric combinations following unfolding of the subunits by treatment at low pH; thus, this specific proteolytic modification does not interfere with the ability of wheat-germ aldolase subunits to refold and to establish precise subunit-subunit recognition in vitro.

Carboxypeptidases↗
PubMed1991

Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding.

The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied. The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS. The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect. Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition. However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS. These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A. (1990) J. Biochem. 108, 572-578), might be involved in the active site or be located in the vicinity of the active site. The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.

Bacteria↗
PubMedDate not supplied

Relationship between enzymatic activity loss and post-translational protein modification in aging.

During aging, there is a decrease in the activity of many enzymes. The mechanism causing the loss of activity is still not well understood in most cases. We have studied the decrease in the activities of the malic, 6-phosphogluconate dehydrogenase and superoxide dismutase enzymes. The old malic enzyme is about 36% less active than the young enzyme and the old 6-phosphogluconic dehydrogenase enzyme is about 26% less active than the young enzyme. In this paper, some chemical properties of these enzymes are studied. Diethyl pyrocarbonate measurements indicate that the old malic enzyme has 1 histidine residue less than the young malic enzyme. Moreover, the treatment of the young malic enzyme with ascorbate for 15 min produces the loss of 36% of enzymatic activity and the loss of 1.2 histidine residues. 2,4,6-trinitrobenzenesulfonic acid measurements indicate that the old 6-phosphogluconate dehydrogenase enzyme has 11 lysine residues less than the young 6-phosphogluconate dehydrogenase enzyme. The proteolysis with trypsin produces more peptides in the young 6-phosphogluconate dehydrogenase enzyme than in the old one. However, similar numbers of peptides were produced when endoproteinase Arg-C was used in both enzymes, young and old 6-phosphogluconate dehydrogenase. Moreover, the treatment of young 6-phosphogluconate dehydrogenase enzyme with ascorbate for 15 min produces the loss of 8 lysine residues. These results suggest that during aging the modification of histidine residue could be involved in the loss of malic enzyme activity, and the modification of lysine residues could be involved in the loss of 6-phosphogluconate dehydrogenase activity. These results could also suggest that the modification of histidine and lysine residues during aging could be produced by oxidation. This could be a general process in aging, with an increase in the oxidation of many proteins. The relevance of this process in the aging effects must be related to the kind of proteins that are susceptible of oxidation and that this oxidation affects their enzymatic or biological function. We have also studied other enzymes one of which is the superoxide dismutase enzyme involved in the protection against oxidative damage. Our results are similar to those described for malic enzyme. In the latter case, the failure to measure one of the histidines in the Cu/Zn SOD is due to a chemical modification, probably caused by oxidation of the residue.

Journal Article↗
PubMedDate not supplied

Age related modifications of soluble proteins in various organs of male garden lizard, Calotes versicolor.

The oxidative modification of proteins measured as carbonyl derivatives increased with advancing age in the liver of male garden lizard. The same parameter did not show a significant change in other organs (brain, heart and kidney). Based on the observations in both homeotherms (mammals) and poikilotherms (insect and reptile), the in vivo oxidative modification of cellular proteins appears to be the most common mechanism leading to accumulation of altered proteins during aging. However, the degree of modifications may vary among the tissues/organs of a species. On the other hand the variations may also account for the role of modification of proteins in the pattern of aging in different species.

Journal Article↗
PubMed1991

Chemical modification studies of Artocarpus lakoocha lectin artocarpin.

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.

Amino Acids↗
PubMed1993

Kinetic study of the addressed modification by hemin derivatives of oligonucleotides.

Kinetics of oligonucleotide pd(TGAATGGGAAGA) modification by a hemin derivative of the complementary oligonucleotide pd(TTCCCATT) in the presence of hydrogen peroxide was investigated. The treatment of experimental data permitted to evaluate the association and rate constants at 25 degrees C: Kx = (3.40 +/- 0.38) x 10(5) M-1 (association constant of the reagent with the target), kd = 152 +/- 6 M-1 min-1 (degradation constant of the hemin group of the reagent in a parallel reaction), ko = 51.0 +/- 1.7 M-1 min-1 (target modification constant in the reactive duplex). The modification of DNA is incomplete due to competition of the modification reaction with the degradation of the hemin group of the reagent in a parallel reaction.

Base Sequence↗
PubMed1986

Effect of limited modification of amino groups on the reactivity of human factor Xa.

The basis of the specificity of human coagulation factor Xa has been probed with a reagent that reacts with nucleophiles, N-succinimidylpropionate. At pH 8.0 and 0.25 mM N-succinimidylpropionate, 0.4 microM factor Xa lost approx. 90% of its activity toward prothrombin in 4 min. The decay was first-order, k = 0.64 min-1, which increased to 0.98 min-1 in 1 mM Ca2+, and the dependence of k upon pH was consistent with primary amines being the target. The rate of modification was unaffected by the presence of a tetrapeptide substrate during modification; likewise, activity toward a tripeptide p-nitroanilide was unaltered during exposure of factor Xa to N-succinimidylpropionate with or without Ca2+. In addition, inhibition by antithrombin III was retained with a somewhat enhanced rate after modification; however, the acceleration of this by heparin was significantly less. Kinetic determination of the number of residues modified gave a reaction order of 2.0, while reaction with N-succinimidyl[3H]propionate yielded labeled factor Xa containing 1.0 mol N-succinimidylpropionate/mol factor Xa and 50% normal clotting activity, or 2.0 mol N-succinimidylpropionate/mol and 1% activity, respectively. Thus, one nucleophilic group is required for the reaction of factor Xa with prothrombin but not for the hydrolysis of peptides or recognition of antithrombin III. The decay of clotting activity of the factor X zymogen in N-succinimidylpropionate was much slower though still Ca2+-dependent. Conversely, the reaction of a related compound--N-succinimidyl(4-hydroxyphenyl)propionate or Bolton-Hunter reagent--with factor Xa broadly resembled that of N-succinimidylpropionate but the decay curves indicated more complex kinetics. Therefore, the target groups vary in their accessibility to modification according to the structural characteristics of both the protein and the reagent.

Antithrombin III↗
PubMed1991

Rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase is locked in the asymmetric state by chemical modification of a single arginine per subunit.

Modification of a single arginine residue per subunit of rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase by 2,3-butanedione converts the enzyme into the form which retains 5-7% of the original activity and manifests cooperative properties that are absent in the native enzyme. It exhibits half-of-the-sites reactivity towards the natural substrate D-glyceraldehyde-3-phosphate. Titration of the modified enzyme with DTNB reveals only two instantaneously reacting SH groups, the total amount of SH groups approaching nine per tetramer. In the presence of 8 M urea, an additional seven SH groups become accessible to DTNB. This suggests that the arginine modification imposes some conformational constraints which affect the microenvironment of the active site cysteine residues in two subunits of the tetramer. The changes do not influence the interaction between the essential cysteine residue and NAD+ which is responsible for the change transfer complex formation, since the molar extinction coefficient of the apoenzyme-NAD+ complex, epsilon 360, was not altered upon the arginine modification. The native and close to four in the case of the native enzyme and about three with the modified one. The apparent pK values of Cys-149 within the functioning active centers of the tetramer were determined from the pH profiles of the inactivation rates in presence of iodoacetamide. The apparent pKa of the essential thiols was found to change upon enzyme modification from 9.44 to 10.07 in the apoenzyme and from 9.17 to 9.36 in the holoenzyme. The apparent pKa of the arginine residue determined from the pH dependence of the inactivation rate was equal to 9.0 and did not change upon apo-holo enzyme transition.

Animals↗
PubMed1992

The efficacy of pharmacokinetic monitoring and dose modification of etanidazole on the incidence of neurotoxicity: results from a phase II trial of etanidazole and radiation therapy in locally advanced prostate cancer.

Fifty-four patients have been entered on a Phase II trial to study the efficacy of etanidazole (ETA) for locally advanced prostate cancer. The primary goal was to study the incidence of and time to a complete response for patients receiving ETA and radiation therapy. The secondary goal was to prospectively evaluate the utility of pharmacokinetic monitoring and dose-modification of the incidence and severity of the dose-limiting peripheral neurotoxicity. Within a constant radiation therapy regimen, the dose of ETA was either (a) unmodified (2 g/m2, 3 times weekly for 17 doses); (b) altered by a schedule modification of either number of doses or dose adjustment; or (c) individualization of single dose size so that the total number of doses (19 doses) were maintained but the single dose size was adjusted to keep the total AUC of plasma concentration versus time to less than 40 mM-hr. Sufficient efficacy data are not yet available. The use of drug dose modification has reduced the incidence of neurotoxicity from (a) unmodified: 17/26 = 65% (1 grade II); (b) schedule adjustment: 5/9 = 55% (no grade II); and (c) individualized dose modification: 1/19 (no grade II) = 6%. The minimum number of time points needed to accurately assess the AUC will be determined. Pharmacokinetic monitoring will be important in the use of ETA so that drug underdosing can be avoided while minimizing the risk of serious neurotoxicity.

Adenocarcinoma↗
PubMed1982

Expression of the Eco RI restriction-modification system and the construction of positive-selection cloning vectors.

The genes encoding the Eco RI restriction-modification (R/M) system have been separately cloned onto compatible plasmids. We have shown that the Eco RI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in Eco RI modification activity at higher temperatures. Insertion of transcriptional terminators into the restriction gene had no detectable effect on Eco RI modification activity. This strongly suggests that a separate promoter exists for the methylase gene. Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene. The temperature-sensitive Eco RI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, Bg/II, or PstI sites inactivates the Eco RI restriction gene and permits the hybrid plasmid to survive at 37 degrees C. The temperature-sensitive modification activity of these vectors should also facilitate the introduction of Eco RI linkers into DNA cloned in this way.

Base Sequence↗
PubMedDate not supplied

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis.

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.

Bacillus subtilis↗