Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “modification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources

Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean.

In the literature, the reports on the effects of a genetically modified (GM) diet are scanty and heterogeneous; in particular, no direct evidence has so far been reported that GM food may affect human or animal health. Hepatocytes represent a suitable model for monitoring the effects of a GM diet, the liver potentially being a primary target. In a previous study, we demonstrated that some modifications occur in hepatocyte nuclei of mice fed on GM soybean. In order to elucidate whether such modifications can be reversed, in the present study, 3 months old mice fed on GM soybean since their weaning were submitted to a diet containing wild type soybean, for one month. In parallel, to investigate the influence of GM soybean on adult individuals, mice fed on wild type soybean were changed to a GM diet, for the same time. Using immunoelectron microscopy, we demonstrated that a one-month diet reversion can influence some nuclear features in adult mice, restoring typical characteristics of controls in GM-fed animals, and inducing in control mice modifications similar to those observed in animals fed on GM soybean from weaning. This suggests that the modifications related to GM soybean are potentially reversible, but also that some modifications are inducible in adult organisms in a short time.

Aging↗

[Sexual failure expectation syndrome and the modification of behavior].

Modifications of behavior were examined in 105 men with sexual failure expectation syndrome. Such modifications were revealed in 77 persons. The determinants and sources of behavioral modifications were defined. It has been shown that phenomenology of behavioral modifications is uncertain, being different as regards the complexity and degree of realization. Different variants of behavioral modifications are described: defense, compensatory, controlling oneself, by the detention type, manipulative, and combined. It is recommended that they be taken into consideration with a purpose of forming in patients of constructive behavioral programs.

Adult↗

Carnosine inhibits modifications and decreased molecular chaperone activity of lens alpha-crystallin induced by ribose and fructose 6-phosphate.

PURPOSE: Alpha-crystallin, a major structural protein in the lens, prevents heat- and oxidative stress-induced aggregation of proteins and inactivation of enzymes by acting as a molecular chaperone. Modification of alpha-crystallin by some posttranslational modifications results in conformational changes and decreases in chaperone activity, which may contribute to cataractogenesis in vivo. Carnosine (beta-alanyl-L-histidine), an endogenous histidine dipeptide, prevents protein modifications including glycation and oxidation. The purpose of this study was to further explore whether carnosine can protect alpha-crystallin against glycation by a sugar and a sugar phosphate, and in particular to find whether it can protect against its decreased chaperone activity. Additionally, we investigated whether carnosine could directly react with a sugar and a sugar phosphate. METHODS: Bovine lens alphaL-crystallin was separated by size-exclusion chromatography on a Sephacryl S-300 HR column. alphaL-crystallin was incubated with different concentrations of fructose 6-phosphate (F6P) and ribose with or without carnosine for different times. The chaperone activity of alphaL-crystallin was monitored using the prevention of thermal aggregation of betaL-crystallin. The modified alphaL-crystallin was examined by SDS-PAGE and fluorescence measurements. The absorbance spectra of solutions of carnosine and sugars were investigated. RESULTS: Carnosine inhibited the crosslinking of alphaL-crystallin induced by F6P and ribose in a dose- and time-dependent manner. It protected alphaL-crystallin against its decreased chaperone activity induced by 100 mM F6P during four days incubation, but not against ribose-induced change. Control alphaL-crystallin gave 96% protection against aggregation of betaL-crystallin after four days incubation, but only 85% protection was achieved in the presence of F6P, rising to 96% (p=0.0004) in the presence of carnosine. After more extensive modification by sugar and a sugar phosphate, there was no significant protective effect of carnosine on alphaL-crystallin cross-linking or chaperone activity. The tryptophan fluorescence of modified alphaL-crystallin was remarkably decreased in the presence of F6P and ribose. However, the decrease was less when 50 mM carnosine was present during eight days incubation with F6P. Carnosine did not maintain the fluorescence when ribose was used. The nontryptophan fluorescence was increased with a shift to longer wavelengths in a time-dependent manner. Carnosine readily reacted with F6P and ribose thereby inhibiting glycation-mediated protein modification as revealed electrophoretically. The increased absorbance was time-dependent, suggesting adducts may be formed between F6P, ribose, and carnosine. CONCLUSIONS: This is the first report showing that carnosine can protect the chaperone activity of alpha-crystallin. This chaperone may protect against cataractous changes. In addition to demonstrating the effects of carnosine on prevention crosslinking, our studies also bring out important evidence that carnosine reacts with F6P and ribose, which suggests carnosine's potential as a possible nontoxic modulator of diabetic complications.

Animals↗

[Epigenetic modification in human leukemia].

Epigenetic modification, which involve DNA methylation, RNA-associated silencing and histone modification, is implicated in cell proliferation, differentiation, survival, apoptosis and malignant transformation. Some leukemogenesis has been shown to be aberrance of epigenetic modification. This paper discussed the potential causes of some of leukemias correlating with the methylation of cell cycle regulation genes, small interference RNA and modification abnormality of histone after translation. The study on epigenetic modification abnormality of leukemia cells provides a new strategy for treatment of leukemia.

DNA Methylation↗

Surgical modifications in bladder neck reconstruction and vesicourethral anastomosis during radical retropubic prostatectomy to reduce bladder neck contractures.

PURPOSE: We describe surgical modifications in radical retropubic prostatectomy (RRP) which have significantly reduced the incidence of bladder neck contractures (BNC). MATERIALS AND METHODS: Between March 1994-July 2005, 977 men underwent a RRP by a single surgeon. Group I comprised 548 patients operated upon July 1994-December 1999, without the modifications described below. Group II comprised 429 men operated upon January 2000-December 2004, with the following surgical modifications: 1) reconstruction of the bladder neck (BN) to a diameter of 28 French; 2) placement of the posterior (6 o'clock) vesicourethral suture on mild traction before placing this suture into the bladder, allowing inspection and, if necessary, replacement of any of the previously placed sutures; 3) bladder displacement when tying the vesicourethral sutures which allows the sutures to be tied under direct vision and prevents incorporation of extraneous tissue. Data were retrospectively analyzed. RESULTS: Demographic data were comparable between groups. In group I, 31/548 (5.7%) developed a BNC compared to 1/429 (0.2%) in Group II, p < 0.001. Urinary continence (no pads/maximum of one light pad for security in 24 hours) at 12 months in the 32 patients who developed a BNC was worse when compared to patients who did not develop a BNC (58% versus 80%, p = 0.003). After excluding the patients who developed a BNC, continence rates were comparable between both groups. The positive margin rate at the BN was not adversely affected by these modifications. CONCLUSIONS: Simple, easily applied modifications to the management of the BN and vesicourethral anastomosis can substantially reduce the incidence of BNC.

Adenocarcinoma↗

[Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate].

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.

Amino Acid Sequence↗

Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase.

Three histidine residues of bovine adrenodoxin, His-10, His-56, and His-62, were modified with diethyl pyrocarbonate. The order of the modification among the three histidines were monitored by measuring the proton NMR spectra. The modified adrenodoxin exhibited reduced affinity for adrenodoxin reductase as determined in cytochrome c reductase activity. In the presence of cholesterol, the modified adrenodoxin induced a high spin form of cytochrome P-450scc on complex formation in the same manner as native adrenodoxin. The spectral titration showed that adrenodoxin modified with diethyl pyrocarbonate exhibited a 5-fold higher Kd value than that of native adrenodoxin. These effects of the modification of adrenodoxin on the affinities for the redox partners were not proportional to the number of modified histidines determined by the optical absorbance change at 240 nm. Modification of adrenodoxin up to 2 histidine residues did not affect the affinity for the redox partners, but further modification on the third one resulted in an increase of apparent Km in cytochrome c reductase activity by 2-fold and of Kd for cytochrome P-450scc by 5-fold. The 1H NMR spectra of the modified adrenodoxin unequivocally demonstrated that histidine residues at His-10 and His-62 reacted more readily with diethyl pyrocarbonate than His-56 did, indicating that modification of His-56 was responsible for the reduction of binding affinities of adrenodoxin for redox partners. These results are consistent with the proposal that the residue of His-56 in adrenodoxin has an essential role in the electron transfer mechanism where adrenodoxin functions as a mobile shuttle.

Adrenodoxin↗

Selective molybdate-directed covalent modification of sulfhydryl groups in the steroid-binding versus the DNA-binding domain of the glucocorticoid receptor.

Hydrogen peroxide produces all of the effects on glucocorticoid receptors that are produced by molybdate, including stabilization of the receptor 90-kDa heat shock protein (hsp90) complex (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). When the glucocorticoid receptor is exposed simultaneously to molybdate and peroxide at concentrations that are optimal for receptor stabilization if each agent is present alone, there is an irreversible loss of steroid binding activity. The effect is accompanied by a covalent modification of the receptor, which is demonstrated by an increase in its apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of the receptor with the sulfhydryl-modifying reagents methyl methanethiosulfonate or N-ethylmaleimide prevents covalent modification, suggesting that cysteine moieties are the site of attack. The covalently modified receptor can still bind to DNA. Molybdate-peroxide treatment does not covalently modify the 15-kDa tryptic fragment containing the DNA-binding domain and 11 of the 20 cysteine moieties in the receptor. However, the 27-kDa tryptic fragment, which contains the steroid-binding domain and 5 cysteines, is covalently modified. The 27-kDa tryptic fragment is covalently modified by the molybdate-peroxide combination when [3H]dexamethasone 21-mesylate is covalently bound to Cys-644. This leaves some combination of 4 cysteines in the steroid-binding domain (628, 649, 671, and 742) as the modified groups. These modifications occur in a region of the receptor that is known to contain its sites of interaction with both hsp90 and molybdate, with the latter having a well-established avidity for sulfur. These observations raise the possibility that the covalent modification caused by the molybdate-peroxide combination represents a modification of sulfur ligands involved in molybdate stabilization of the receptor.

Affinity Labels↗

Binding of heparin to human antithrombin III activates selective chemical modification at lysine 236. Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of antithrombin III.

A new water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC), was used to identify lysine residues of antithrombin III which participate in the binding of heparin. Antithrombin, modified with S-DABITC in the presence and absence of low molecular weight heparin (Mr 5000) was reduced, carboxymethylated, and digested with trypsin. The digest was analyzed by high-performance liquid chromatography and monitored at 465 nm. In the absence of heparin, four major colored peptides (T1, T2, T3, and T4) were identified. When antithrombin was preincubated with heparin (2-fold by weight), followed by S-DABITC modification, the recovery of peptide T4 remained unchanged, but the recoveries of T1, T2, and T3 were reduced by 93, 86, and 98%, respectively. In addition, a new colored peptide, TA, appeared. Amino acid sequencing of peptides T1, T2, T3, and TA localized S-DABITC modification sites as Lys-136, Lys-125, Lys-107, and Lys-236, respectively. Thus, binding of heparin to human antithrombin diminished S-DABITC modification at Lys-107, Lys-125, and Lys-136, but at the same time enhanced S-DABITC modification at Lys-236. This phenomenon was further characterized by varying the molar ratio of heparin/antithrombin (from 0.04 to 20). The shielding of Lys-125 and Lys-136 was inversely proportional to the activation of Lys-236. At a heparin/antithrombin molar ratio of 1, the extent of shielding of Lys-125 and Lys-136 and the unmasking of Lys-236 were 25-33%. This shielding-unmasking effect correlated with enhanced antithrombin inhibition of thrombin. We conclude that Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of human antithrombin and that binding of heparin to antithrombin causes a conformational change of antithrombin that leads to the exposure of Lys-236 for S-DABITC modification.

Amino Acid Sequence↗

Inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyl[3H]inosine is accompanied by modification of tyrosine 345 in a single beta subunit.

The inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoylinosine (FSBI) proceeds with pseudo-first order kinetics. The rate of inactivation increased from pH 7 to 9 revealing a pKa of about 8.2. When a tryptic digest of the enzyme which had been inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine ([3H]FSBI) was submitted to reversed phase high pressure liquid chromatography, a single major peak of radioactivity, T1, was resolved. Amino acid sequence analysis of purified peptide fragments derived from T1 showed that the modification of beta-Tyr-345 is responsible for inactivation of the enzyme. Complete inactivation of the enzyme by [3H]FSBI is estimated to proceed with modification of 0.8 mol of beta-Tyr-345/mol of enzyme. Another notable observation is that inosine triphosphatase (ITPase) activity catalyzed by F1 from bovine heart mitochondria is much more sensitive to inactivation by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) than is ATPase activity. Whereas complete inactivation of ATPase activity by FSBA has been shown to proceed with the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the beta subunit (Bullough, D. A., and Allison, W. S. (1986) J. Biol. Chem. 261, 5722-5730), it is shown here that complete inactivation of ITPase activity by FSBA is accompanied by modification of these residues in only one copy of the beta subunit. Inactivation of both the ATPase and ITPase activities of the enzyme by FSBI proceeds with modification of Tyr-345 in a single copy of the beta subunit.

Adenosine↗

Chemical modification of the bifunctional regulatory protein of maize leaf pyruvate,orthophosphate dikinase. Evidence for two distinct active sites.

The active site(s) of the bifunctional regulatory protein of pyruvate,orthophosphate dikinase catalyze(s) the Pi-dependent activation (dephosphorylation) and ADP-dependent inactivation (phosphorylation) of maize leaf dikinase. The chemical modification studies of the regulatory protein active sites presented in this paper are interpreted as showing the two sites to be physically distinct. Pyridoxal 5'-phosphate and 2-nitro-5-thiocyanatobenzoate (NTCB) selectively inhibit the dikinase activating site, which is protected by the nonprotein substrate, Pi. Phenylglyoxal blocks both the activation and inactivation sites; the former is protected selectively by Pi and the latter by both the nonprotein substrate, ADP, and Pi. The Pi that protects the inactivation site is distinct from the activation substrate. Inhibition studies show Pi to be a parabolic competitive inhibitor of the ADP-dependent inactivation of dikinase, implying that besides substrate Pi, a second phosphate also binds to the regulatory protein. The above chemical modifications are not mutually exclusive; neither NTCB, 5,5'-dithiobis-(2-nitrobenzoate), nor pyridoxal 5'-phosphate blocks subsequent modification of the activation site by phenylglyoxal. Similarly, prior modification with NTCB does not affect modification by pyridoxal 5'-phosphate.

Adenosine Diphosphate↗

The heparin binding site of human antithrombin III. Selective chemical modification at Lys114, Lys125, and Lys287 impairs its heparin cofactor activity.

Heparin binds to human antithrombin III and accelerates its inhibitory activity in the blood coagulation system. Previous reports (Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505; Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938) have shown that selective chemical modification of a limited number of lysine residues in antithrombin III causes drastic loss of its heparin cofactor activity. We have performed chemical modification of antithrombin III with trinitrobenzene sulfonic acid in order to determine the location of these lysine residues. When antithrombin III was treated with 100 M excess of trinitrobenzene sulfonic acid for 10 min, about 3.2 mol of amino group per mol of antithrombin III were modified. The heparin cofactor activity dropped to about 25%, whereas the progressive inhibitory activity (in the absence of heparin) remained essentially intact (about 95%). The modified amino groups were identified to be Lys114 (75%), Lys125 (94%), and Lys287 (96%). These results were obtained by comparing and analyzing the cyanogen bromide fragments derived from native antithrombin III and the 10-min modified antithrombin III. When antithrombin III was pretreated with heparin, followed by trinitrobenzene sulfonic acid modification, the extent of modification at Lys114 and Lys125 decreased from 75% and 94% to 20% and 40%, respectively, whereas the modification at Lys287 remained nearly quantitative (greater than 95%). Based on these results, we conclude that Lys114 and Lys125 are essential for the heparin cofactor activity of human antithrombin III.

Amino Acids↗

Modification of the fatty acid composition of cultured human fibroblasts.

The fatty acid composition of human skin fibroblasts grown in 10% dialyzed fetal calf serum can be modified considerably by adding supplemental fatty acids to the culture medium. The degree of modification was dependent on the concentration of added fatty acid over the range tested, 2.5 X 10(-5) to 1 X 10(-4) M. At the higher concentration, the extent of the modifications was as those which can be produced in nonhuman or malignant cell lines. Although the greatest changes were produced in the neutral lipid fraction, the cellular phospholipids also exhibited appreciable modifications. The phospholipids isolated from a microsomal fraction prepared from the cell homogenate exhibited similar changes in fatty acyl composition. These findings indicate that the human fibroblast can tolerate considerable variability in fatty acid composition, even in membrane phospholipids. The triglyceride content of the cells increased when they were grown in the presence of added fatty acids, but the phospholipid and cholesterol content remained unchanged. Growth was not affected by either oleic or linoleic acids, but it was reduced up to 50% when palmitic linolenic, or arachidonic acid was added in concentrations of 5 X 10(-5) M or above. Extensive modifications in phospholipid fatty acid composition also were produced in confluent monolayers of these fibroblasts. This suggest that some membrane lipid turnover occurs even when the cultures are not rapidly growing. Fatty acid modifications also were produced in the commercially available IMR-90 strain of human lung fibroblasts, suggesting that the ability to tolerate considerable differences in fatty acid composition is not a special property of the skin fibroblast line that was isolated locally.

Cell Line↗

[Cellular actions of inhibitors of chemical carcinogenesis: modifications of syntheses of prostaglandins and differentiation of myeloid leukemia cells].

Although various substances are involved in modification of chemical carcinogenesis, prostaglandins have been known to play an important role in the modification of the carcinogenesis. In this report, recent advances in the modification of chemical carcinogenesis by prostaglandins are reviewed. In addition, recent experimental results on the modification of cell differentiation by prostaglandins and some inhibitors of chemical carcinogenesis are described. Some inhibitors of chemical carcinogenesis are found to modify differentiation of mouse myeloid leukemia cells (M1) into macrophages and granulocytes by perturbation of syntheses of prostaglandins. These experimental results suggest that the modification by the inhibitors of carcinogenesis of differentiation of cells by perturbation of syntheses of prostaglandins is involved in the mechanisms of inhibition of chemical carcinogenesis.

Animals↗

[Effect of progressive chemical modification on the activity and thermal stability of soluble and immobilized glucoamylase].

The chemical modification of glucoamylase from Aspergillus niger with acryloylchloride has been studied. It was shown that the first 60--70% of the total amount of available amino groups of the enzyme readily interact with the reagent: the activity and thermal stability of the resulting soluble acryloylglucoamylase are only slightly less than those of the native enzyme. The modification of the remaining 30% of the total amount of amino groups is less intensive and results in a sufficient loss of activity (6 times at 92% modification) and thermal stability (3,5 times at the same degree of modification) by the enzyme. The multipoint immobilization of glucoamylase by mutual covalent attachment of its acryloyl derivative to polyacrylamide gel also causes destabilization of the enzyme. The resulting increase of the number of the enzyme--support binding points leads to a progressive decrease of the enzyme thermal stability as compared to native glucoamylase despite a slight increase in stability (up to 1,5 times) as compared to soluble acryloylglucoamylase. It was shown that destabilization of glucoamylase at 65 degrees due to chemical modification is more pronounced than its negligible stabilization due to the multipoint fixation of glucoamylase on a high polymer support.

Acrylates↗

Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues.

The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.

Adenylyl Cyclases↗

Pharmacology and toxicity of intracarotid adriamycin administration following osmotic blood-brain barrier modification.

The effect of reversible blood-brain barrier modification on the delivery of Adriamycin to the brain was studied in a rodent and canine model. Pharmacokinetic and physiological studies were done in these animals after a wide range of doses of Adriamycin (0.1 to 1.0 mg/kg) were administered into the carotid artery following osmotic barrier modification with mannitol. In the absence of barrier modification, no immunoreactive Adriamycin was detected in the cerebrum; whereas, following barrier modification, up to 4.5 micrograms of drug and/or metabolites per g of brain were found. Optimum tissue levels of Adriamycin and metabolites were achieved following barrier modification when the drug was administered by either bolus or slow continuous (15-min) infusion. Immunoreactive drug was identified in brain for up to 6 hr after administration. Significant functional neurotoxicity occurred at all dose levels, even at 0.1 mg/kg, a level at which Adriamycin concentration in the brain was below the level of detectability. Neuropathological examination revealed the presence of necrosis and hemorrhagic infarcts. Thus, these pharmacological and toxicity studies suggest that Adriamycin (or its metabolites) may produce significant clinical neurotoxicity when even small amounts penetrate the blood-brain barrier.

Animals↗

Structure-function relationship in Escherichia coli initiation factors. Identification of a lysine residue in the ribosomal binding site of initiation factor by site-specific chemical modification with pyridoxal phosphate.

Incubation of Escherichia coli initiation factor 3 (IF3) with pyridoxal phosphate (PLP) followed by reduction with sodium borohydride resulted in the selective modification and inactivation of this protein. The ribosomal-binding site (RNA-binding site) of IF3 is the target of PLP modification, since (a) the phosphate residue of PLP is required for inactivation; (b) RNA as well as synthetic polynucleotides (especially guanine-containing one) protect IF3 from inactivation; and (c) 30 S, but not 50 S ribosomal subunits, protect IF3 from PLP modification and from inactivation. The incorporation of PLP into IF3 occurred exclusively at lysine residues by reduction of the Schiff bases yielding epsilon-(5'-phosphopyridoxyl)lysine. The PLP-modified lysines were identified by amino acid analysis and sequencing of the PLP-modified peptides. Out of the 20 lysines of the factor, only Lys 2, Lys 5, Lys 99, Lys 112, Lys 166, and an unidentified Lys of the central cluster of the molecule (Lys 86, 87, 90, 91, 96) were found to be modified to varying degrees. The incorporation of 3 to 4 mol of PLP/mol of IF3 is accompanied by a substantial (greater than or equal to 80%) inactivation of this protein; the loss of activity follows apparent first order kinetics, and the inactivation results from the modification of just 1 Lys residue. This essential Lys residue was identified by various criteria to be Lys 112. The identification of an "active region" in the IF3 molecule is emerging from this as well as from other chemical modification studies.

Amino Acids↗