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PubMed1997

Nitric oxide-induced modification of glyceraldehyde-3-phosphate dehydrogenase with NAD+ is not ADP-ribosylation.

One biological effect of nitric oxide (NO) has been believed to be exerted through induction of the ADP-ribosyltransferase activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Though this notion is based on the finding that NO increases the auto-ADP-ribosylation of GAPDH, controversial data have also been reported. To determine whether or not NO really activates ADP-ribosylation, we re-examined the NO-induced modification of GAPDH with NAD+. GAPDH was modified equally with [adenosine-14C]NAD+ and [carbonyl-14C]NAD+, indicating that the glycoside bond of NAD+ between ADP-ribose and nicotinamide is intact. The release of nicotinamide from NAD+ was not evident during incubation of GAPDH with [carbonyl-14C]NAD+. Thus, the modification of GAPDH is apparently not ADP-ribosylation. In addition, we found that basal and glyceraldehyde-3-phosphate-induced modifications of GAPDH, both of which have also been explained as ADP-ribosylation, were not ADP-ribosylation, and that the modification of GAPDH in the absence and presence of NO or GA3P was distinct in the dithiothreitol effect or resistance to HgCl2.

Adenosine Diphosphate Ribose↗

PubMed1988

Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli.

Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.

Binding Sites↗

PubMed1988

Prediction of in-vivo modification sites of proteins from their primary structures.

In order to make better use of the information contained in rapidly expanding amino acid sequence data, a new method to predict various modification sites of proteins from their primary structures is presented. It is also applicable to the prediction of other functional sites in proteins. Here we show the examples of N-glycosylation and serine/threonine phosphorylation sites. The method is essentially an elaboration of consensus sequence pattern matching based on stepwise discriminant analysis. The occurring amino acids near a potential modification site are represented by six numerical values which reflect various properties of amino acids. Longer-range effects around these sites are also considered. The stepwise procedure enabled us to automatically select effective features for discrimination. A computer program with our method first identifies potential modification sites by a sequence pattern, NX(S/T) for N-glycosylation or (S/T) for phosphorylation, and then decides by discriminant analysis whether a potential site is likely to be a true modification site. The prediction accuracy in the second step of discrimination was about 60% for glycosylation sites and about 80% for phosphorylation sites.

Amino Acid Sequence↗

PubMed1975

The modification of sulfhydryl groups of glutamine synthetase from Bacillus stearothermophilus with 5, 5'-dithiobis(2-nitrobenzoic acid).

The SH groups of glutamine synthetase [EC 6.3.1.2] from Bacillus stearothermophilus were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) in order to determine the number of SH groups in the molecule as well as the effect of the modification on the enzyme activity. Three SH groups per subunit were detected after complete denaturation of the enzyme with 6 M urea, one of which was essential for the enzyme activity in view of its reactivity with 5, 5'-dithiobis(2-nitrobenzoic acid) on addition of MgCl2 with loss of the activity. The CD spectra of the modified enzyme in the near ultraviolet region changed from that of the native enzyme, indicating that aromatic amino acid residues were affected by modification of the SH group. The fluorescence derived from tryptophanyl residue(s) was quenched depending on the extent of modification of the SH group, suggesting that the tryptophanyl residue(s) was located in the proximity of the SH group. The thermostability of the enzyme was remarkably decreased by modification of the SH group.

Binding Sites↗

PubMed1976

Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin.

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.

Actins↗

PubMed1983

Modification of a major ribonuclease from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

In order to investigate the role of carboxyl groups of a base non-specific ribonuclease from Aspergillus saitoi [EC 3.1.27.1] (RNase M, molecular weight 36,000), the modification of RNase M with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide(CMC), was studied. The inactivation of RNase M proceeded almost linearly with the incorporation of about 9.5 CMC moieties. The peptide backbone structure of the modified RNase M was practically the same as that of the native RNase M, as assessed from the CD spectra in the region of 200-250 nm. In the presence of competitive inhibitors, adenosine, and cytidine, inactivation of RNase M by CMC was partially inhibited. In the presence of cytidine (1 M), the modification of about 4 carboxyl groups of RNase M proceeded with a slight loss of enzymatic activity (ca. 20%). Further modification inactivated RNase M with the incorporation of ca. 4-5 CMC without any detectable intramolecular peptide bond formation. Therefore, it was concluded that carboxyl groups responsible for enzymatic activity were included among these carboxyl groups protected by cytidine. The logarithm of the half-live of the inactivation of RNase M by CMC was a linear function of log[CMC] with a slope of minus one, indicating that at least one carboxyl group among the modified ones may be essential for catalysis. The digestion of CMC-modified RNase M with carboxypeptidase A eliminated the carboxyl terminal group from the site of CMC modification.

Amides↗

PubMed1984

Modification of glucoamylases from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

To investigate the role of carboxyl groups of glucoamylases [EC 3.2.1.3] from a Rhizopus sp. (Gluc1 and Gluc2), the modification of Gluc1 and Gluc2 with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), was studied. The inactivation of Gluc1 proceeded with the incorporation of about 3 CMC moieties. In the presence of maltose, the modification of about 2.2 carboxyl groups of Gluc1 proceeded with a slight loss of enzymatic activity. In the re-modification of Gluc1 modified in the presence of maltose, Gluc1 was inactivated by further modification of about 1.3 carboxyl groups. Therefore, one carboxyl group, which was protected by maltose, was thought to be a crucial one. The inactivation of Gluc2 proceeded similarly to that of Gluc1, but the number of CMC moieties incorporated was about one less than in the case of Gluc1. Thus, it was suggested that one of the reactive carboxyl groups of Gluc1 was located in the N-terminal part of Gluc1, which is deficient in Gluc2. From the results of kinetic studies on CMC-modified Gluc1, it was suggested that the hydrolysis mechanism of malto-oligomers differs somewhat from that of PNPG.

CME-Carbodiimide↗

PubMed1985

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate.

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.

Adenosine Monophosphate↗

PubMed1988

Cofactors in and as posttranslational protein modifications.

A symposium at the FASEB meeting in Las Vegas in May 1988 will be devoted to the role of cofactors (vitamins, coenzymes, prosthetic groups) in and as posttranslational protein modifications; the symposium is part of a thematic focus on metabolic regulation. In planning the symposium, we decided to consider metabolic regulation in its broadest context, which should include both the short-term activity modulations in the life of contemporary organisms and the adaptations of special molecular strategies over evolutionary time. We further decided to focus the symposium context on the involvement of cofactors both as catalytic participants in and as substrates or end products of posttranslational modifications. As a preview of the actual symposium, the present discussion is an attempt to enumerate cases of cofactor involvement in these different categories: 1) essential nutrients as participants in posttranslational modifications; 2) cofactors as donor substrates in reversible, regulatory modifications; and 3) cofactor incorporation or generation as covalent constituents of proteins. The actual symposium topics are taken from category 1: vitamin C and protein hydroxylation (K. I. Karivikkio) and vitamin K and protein carboxylation (J. W. Suttie) and category 3: biotinylation (H. G. Wood), phycobiliproteins (A. Glazer), and pyruvoyl enzymes (W. Dowhan).

Animals↗

PubMed1978

Modification of the Longmire procedure.

Two modifications of the Longmire procedure of intrahepatic cholangiojejunostomy are reported. The first involves the preoperative placement under fluoroscopic control of a large Teflon catheter in the distal left hepatic duct to facilitate the identification and isolation of the duct at the time of surgery. The second modification is the use of a transhepatic silastic biliary stent positioned by utilizing the Teflon catheter. These modifications make the Longmire procedure technically much easier, and should help insure long-term success in both benign and malignant strictures. A successful case utilizing these modifications is reported.

Adult↗

PubMed1986

The prognostic value of the modifications of the Dukes' C class of colorectal cancer. An analysis of the NSABP clinical trials.

This study was carried out in an effort to resolve the dilemma created by three proposed modifications of the Dukes' C class of colorectal cancer. Each modification is based on a separate prognostic discriminant characterized by: the level of histologically positive nodes, the depth of tumor penetration, and the number of histologically positive nodes. Data were derived from 844 patients with Dukes' C lesions randomized into two prospective clinical trials of the NSABP; the mean time on study was 41 months. Analysis of the three modifications as independent variables without regard for possible confounding effects disclosed that each had a highly significant predictive capacity. When each discriminant was examined, this time adjusting for the contribution of the other two discriminants, the effect attributable to the level of positive nodes was markedly attenuated. Thus, the level of positive nodes provided little information over and above that of depth of tumor penetration and the number of positive nodes. Of the two latter discriminants, although both were significant predictors of survival, the number of positive nodes appeared to be the strongest factor. Using both depth of penetration and the number of positive nodes, a unique Dukes' C subset of patients could be identified with a prognosis at least as good as Dukes' B lesions; this group was characterized by partial tumor penetration and the presence of 1-4 positive nodes. It is concluded that both depth of penetration and the number of positive nodes represent appropriate modifications of the initial Dukes scheme, and one discriminant should not be used to the exclusion of the other. The data raise serious doubts relative to the propriety of newly proposed TNM classification schemes that fail to utilize the number of positive nodes as a predictive discriminant.

Carcinoma↗

PubMed2002

Posttranslational protein modifications: new flavors in the menu of autoantigens.

Perhaps one of the most elusive areas of study in autoimmunity has been identifying the self-antigens that initially trigger the development of autoimmune responses. Recent work in this area has demonstrated that a number of biochemical modifications that arise in proteins after their translation induce autoimmune responses to otherwise ignored self-proteins. This article will describe those autoimmune diseases in which posttranslational modifications may play a role in initiation of disease, as well as identify how these modifications arise and contribute to the breakdown of immune tolerance. Lastly, we will address how posttranslational modifications in self-antigens affect current diagnostic techniques and the development of immunotherapies for autoimmune diseases.

Amino Acids↗

PubMed1994

Environmental stress-induced functional modification of the central benzodiazepine binding site.

The central benzodiazepine binding site mediates the therapeutically relevant pharmacologic actions of benzodiazepine agonists, including reducing anxiety, sedation, muscle relaxation, and antagonism of seizure production. Benzodiazepines potentiate the ability of gamma-aminobutyric acid (GABA) to promote membrane chloride ion conductance by binding reversibly to a distinct site on the GABAA receptor complex. Although the role of this central benzodiazepine binding site in mediating the pharmacologic actions of benzodiazepine agonists has been shown conclusively, its participation in an animal's response to stress is less certain. Data are reviewed consistent with an environmental stress-induced modification of the structure and function of the central benzodiazepine binding site. Modifications show brain anatomic regional selectivity. The mechanisms of these modifications include rapid enzyme-mediated phosphorylations of the GABAA receptor complex and the selective transcription of individual polypeptide subunits. The potential relevance of the data on environmental stress-induced functional modifications of the central benzodiazepine binding site to the development of newer medications and improved understanding of the pathophysiology of stress-related neuropsychiatric disorders is discussed.

Animals↗

PubMed1999

Decrease and structural modifications of phosphatidylethanolamine plasmalogen in the brain with Alzheimer disease.

Several lipid modifications, some of which were attributed to oxidative stress, have been reported in the brains of patients with Alzheimer disease (AD). To evaluate this possibility, all phospholipids and their ether subclasses from the frontal cortex, hippocampus, and the white matter of AD brain were analyzed by high performance liquid chromatography and gas chromatography. The total phospholipid in the frontal cortex and hippocampus decreased on a DNA basis by about 20% and this change was essentially explained by a selective decrease in phosphatidylethanolamine and phosphatidylcholine. The lower content of phosphatidylethanolamine was due to a specific decrease in the plasmalogen subclass. Phosphatidylethanolamine plasmalogen was also the only lipid exhibiting major structural modifications: a significant decrease in polyunsaturated fatty acids and oleic acid as well as a shift of the aldehyde pattern from 18:1 to 18:0. The only modification observed in the other phospholipids was a decrease in oleic acid in diacyl-phosphatidylethanolamine and diacyl-phosphatidylcholine. None of these changes were observed in the white matter. Both the vinyl ether bond of phosphatidylethanolamine plasmalogen and polyunsaturated fatty acids are major targets in oxidative stress; thus, these specific lipid modifications strongly support the involvement of free radicals in the pathogenesis of AD.

Aged↗

PubMed1992

Structural and functional evaluation of modifications in the composite skin graft: cryopreserved dermis and cultured keratinocytes.

Structural and functional aspects of modifications in the composite skin graft consisting of cultured keratinocytes and cryopreserved dermis were determined. Cryopreserved human cadaveric dermis separated from skin by short and mild trypsinization was compared with dermis obtained by prolonged incubation in medium and with fresh dermis obtained by the same methods. All types of dermis were shown to retain normal ultrastructure and topographic organization, as detected by scanning and transmission electron microscope and immunofluorescence analysis. However, in fresh skin, the layers were more firmly attached, mechanical separation was more difficult, and residual epidermis often remained attached to the dermis. Keratinocytes attached better, began replication earlier, and generally reached higher cell numbers when cultured on trypsinized dermis than on medium-treated dermis. The performance of several modifications in the reconstitution and grafting procedures of the composite skin graft after transplantation to athymic mice was examined. Cultured epidermis combined onto trypsinized or medium-treated whole and meshed dermis, dermis pregrafted and allowed to take before transplanting epidermis on top, and keratinocytes grown into multiple epithelia on top of trypsinized meshed or whole dermis prior to grafting. The best grafting results were obtained with an "instant" reconstituted skin model: multiple epithelia grown in vitro combined immediately before grafting onto meshed trypsinized dermis. The transplantation results of this modification were significantly better than those of all the other modifications, including initial growth of keratinocytes into multiple epithelia on top of trypsinized dermis prior to grafting.

Animals↗

PubMed2006

Effect of hepatitis C coinfection on discontinuation and modification of initial HAART in primary HIV care.

OBJECTIVES: To estimate the effect of hepatitis C (HCV) coinfection on time to first occurrence of either discontinuation or modification of initial HAART among previously antiretroviral therapy-naive HIV-infected patients. METHODS: The analysis included antiretroviral therapy-naive patients who initiated HAART prior to November 2003 and were participating in the University of North Carolina Center for AIDS Research, HIV/AIDS observational clinical cohort. The effect of HCV status on time to first occurrence of either HAART discontinuation or modification was assessed using Kaplan-Meier survival estimates and multivariable proportional hazards regression was used to estimate hazard ratios. RESULTS: Of 296 patients initiating HAART, 22% were coinfected with HCV. During a median follow-up of 473 days [interquartile range (IQR), 167-940] from HAART initiation, 104 (35%) patients discontinued and 91 (31%) modified their first regimen. Reasons for discontinuation and modification were comparable by HCV serostatus and included treatment failure (12%), toxicity (41%), and barriers to adherence (47%). The median time to first occurrence of either discontinuation or modification among HCV-infected patients was 401 days (IQR, 128-821), and among HCV-uninfected patients was 493 days (IQR, 204-952) (P = 0.22). After adjustment for baseline demographic and clinical characteristics, the hazard ratio contrasting HCV-infected with HCV-uninfected patients was 1.39 (95% confidence interval, 0.95-2.03; P = 0.09). CONCLUSION: HCV coinfection was only marginally associated with a shorter duration of an initial HAART regimen, suggesting optimization of a first HAART regimen may not appreciably depend on HCV serostatus.

Adult↗

PubMed1980

Streptomyces albus G mutants defective in the SalGI restriction-modification system.

Streptomyces albus G mutants (at least 12 of which were independent) defective in SalGI-mediated restriction (R-) were isolated after mutagenesis. Some of them lacked detectable SalGI activity in cell-free extracts. Some were also partially or completely defective in SalFI-associated modification (M-). Loss of restriction rendered S. albus G sensitive to many phages to which it was normally totally resistant. DNA from one such phage had many SalGI target sites (mean, one site per 1.35 kilobases). A mutant was isolated which was heat-sensitive for growth, apparently because it was restriction-proficient but temperature-sensitive for modification. At a rather high frequency, this mutant generated spontaneous heat-tolerant derivatives which were nearly all R-. Such R- mutants were always M- rather than being temperature-sensitive for modification. In a limited genetic analysis, the determinants of restriction and modification did not recombine with each other, and since there was no reassortment of these phenotypes among the parental output of crosses it appeared that the determinants were located close together on the chromosome.

Bacteriophages↗

PubMed1991

Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.

Alcohol Oxidoreductases↗