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PubMedDate not supplied

Modifying the NH2 and COOH termini of aquaporin-5: effects on localization in polarized epithelial cells.

To reengineer polarized epithelial cell functions directly in situ, or ex vivo in the fabrication of an artificial organ, it is necessary to understand mechanisms that account for polarized membrane sorting. We have used the aquaporins (AQPs), a family of homotetrameric water channel proteins, as model membrane proteins for this purpose. AQP monomers contain six transmembrane-spanning domains linked by five interconnecting loops, with the NH2 and COOH termini residing in the cytosol. AQP5 is localized in the apical membranes of several different epithelia in vivo, and in stably transfected MDCK-II cells grown as a polarized monolayer. We wished to identify a structural region(s) within rat AQP5 (rAQP5) important for apical localization, and to study the MDCK-II cell localization of rAQP5s modified in either their NH2 or COOH terminus. We show that the NH2- terminal region does not play a major role in apical localization as deletion of the NH2 terminus produced a modified rAQP5 construct (AQP5-NT(del)) that was stably expressed and localized primarily to the apical membranes of MDCK-II cells. Attachment of a FLAG epitope to the NH2 terminus of AQP5 (AQP5(flag) construct) also did not perturb apical localization. In addition, we found that the exchange of NH2-terminal regions between rAQP5 and human AQP1 (hAQP1; a nonpolarized AQP isoform) produced a modified rAQP5 construct (AQP5-1NT) and a modified hAQP1 construct (AQP1-5NT), each of which localized as the parental AQP (apically, and to both apical and basolateral membranes, respectively). In contrast, we found that deletion of the COOH terminus resulted in a modified rAQP5 construct (AQP5-CT(del)) that was unstably expressed and localized to intracellular site(s) in MDCK-II cells. Substitution of the COOH terminus of AQP1 with the COOH terminus of AQP5 also produced a construct (AQP1-5CT) transiently expressed in intracellular compartment(s). However, substitution of the COOH terminus of rAQP5 with the COOH terminus of hAQP1 produced a modified rAQP5 construct (AQP5-1CT) that was stably expressed and localized to basolateral membranes, suggesting the loss of an apical targeting/retention signal from rAQP5, the gain of a basolateral targeting/retention signal from hAQP1, or a combination of these two possibilities.

Animals↗
PubMed1990

Characterization of the DNA damage recognized by an antiserum elicited against cis-diamminedichloroplatinum (II)-modified DNA.

A series of in vitro and in vivo studies were performed to characterize DNA damage recognized by an antiserum elicited against DNA modified with cis-diamminedichloroplatinum(II) (cisplatin). Adducts determined by the cisplatin-DNA enzyme-linked immunosorbent assay (ELISA) in human blood cell DNA have been shown to correlate well with positive clinical outcome in testicular and ovarian cancer patients receiving platinum drug-based chemotherapy (Reed et al. (1990) Proc. Natl. Acad. Sci., 84; 5024, and Reed et al. (1988) Carcinogenesis, 9, 1909). DNAs from calf thymus, salmon sperm, pBR322 and synthetic oligonucleotides were modified with cisplatin in vitro before or after specific DNA digestion steps to yield adducted samples of known size and/or chemical composition. These cisplatin modified DNAs were assayed by atomic absorption spectrometry (AAS) to assess absolute platinum content, and by ELISA to determine the antiserum specificity. The antiserum recognizes native cisplatin-modified calf thymus DNA, and native oligonucleotides containing intrastrand cis-Pt (NH3)2-d(pGpG) adducts (Pt-GG) and intrastrand cis-Pt (NH3)2-d(pApG) adducts (Pt-AG). Modified plasmid DNA fragments of varying sizes (down to 309 base pairs) are recognized similarly to cisplatin-modified calf thymus DNA. The antiserum does not cross-react with individual Pt-GG or Pt-AG adducts not bound to DNA. In experiments designed to assess the relationship between adduct measured by ELISA and total platinum bound to DNA as measured by AAS, male and female Sprague-Dawley rats were injected i.p. with cisplatin and a dose response for adduct formation was determined in kidney DNA samples. Values obtained by ELISA were substantially lower than those measured by AAS, and the two were directly related in DNA from kidney tissues of rodents but not in DNA from human nucleated blood cells. In rodent samples the ELISA measured a consistent 0.2% of the total DNA-bound platinum determined by AAS, with a correlation coefficient of 0.91. Among 54 blood cell DNA samples from human patients, which gave measurable adduct values in both ELISA and AAS, the ELISA measured a variable fraction (0.2-33.0%) of the total DNA-bound platinum measured by AAS. We conclude that the cisplatin-DNA ELISA measures a three dimensional lesion in DNA that is formed in direct proportion to total DNA-bound platinum in rat kidney, but that in human biological samples, interindividual variability precludes a relationship that conforms to simple mathematical algorithms.

Animals↗
PubMed1983

Translation of satellite tobacco necrosis virus RNA modified by (not equal to)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is inhibited in a wheat germ cell-free system.

It has been shown that (not equal to)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) modification of rabbit globin mRNA results in inhibition of translational initiation. In order to explore the possibility that modification of the 5' cap structure was responsible for this inhibition, the naturally non-capped mRNA from satellite tobacco necrosis virus (STNV) was reacted with BPDE and translated in a wheat germ cell-free system. The extent of modification was 1.3 and 2.9 BPDE residues/molecule. High performance liquid chromatography of the modified nucleosides from enzymatically hydrolyzed STNV RNA revealed that greater than 90% of the nucleoside adducts were substituted at the exocyclic amino group of guanosine. The translational ability of the lower and higher modified STNV, measured by incorporation of [14C]amino acids into acid-precipitable polypeptides is inhibited by 55% and 63%, respectively. Polyacrylamide gel electrophoretic analyses of the translation products indicate that predominantly full-length coat proteins are synthesized but with the carcinogen-modified STNV the amount is reduced. On the other hand, 80S initiation complex formation is not inhibited as measured by binding of the BPDE-modified STNV to ribosomes and followed by glycerol gradient centrifugation. Under these conditions, aurintricarboxylic acid completely inhibits 80S initiation complex formation in the presence of either modified or native STNV. These results suggest that inhibition of in vitro translation of BPDE-modified STNV, in contrast to that of globin mRNA, is not at the level of initiation complex formation but possibly by premature termination of growing polypeptides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Monoclonal antibodies to a benzo[a]pyrene diolepoxide modified protein.

Monoclonal antibodies have been developed which specifically recognize protein modified by 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). These antibodies have been characterized as to sensitivity and specificity by an enzyme-linked immunosorbent assay. They react with BPDE-I-modified proteins and do not cross-react with non-modified protein. All the antibodies have higher sensitivity toward BPDE-I-tetraols than to BPDE-I-modified protein. They cross-react with BPDE-I-deoxyguanosine, BPDE-I-DNA and BPDE-II-rabbit serum albumin. Because of the high antibody sensitivity for tetraols, an immunoassay was developed for quantitating DNA and protein adducts based on release of tetraols from modified samples. This approach was validated using radiolabeled BPDE-I modified DNA and protein.

Antibodies, Monoclonal↗
PubMed1994

Weakener of white (Wow), a gene that modifies the expression of the white eye color locus and that suppresses position effect variegation in Drosophila melanogaster.

A locus is described in Drosophila melanogaster that modifies the expression of the white eye color gene. This trans-acting modifier reduces the expression of the white gene in the eye, but elevates the expression in other adult tissues. Because of the eye phenotype in which the expression of white is lessened but not eliminated, the newly described locus is called the Weakener of white (Wow). Northern analysis reveals that Wow can exert an inverse or direct modifying effect depending upon the developmental stage. Two related genes, brown and scarlet, that are coordinately expressed with white, are also affected by Wow. In addition, Wow modulates the steady state RNA level of the retrotransposon, copia. When tested with a white promoter-Alcohol dehydrogenase reporter. Wow confers the modifying effect to the reporter, suggesting a requirement of the white regulatory sequences for mediating the response. In addition to being a dosage sensitive regulator of white, brown, scarlet and copia, Wow acts as a suppressor of position effect variegation. There are many dosage sensitive suppressors of position effect variegation and many dosage-sensitive modifiers of gene expression. The Wow mutations provide evidence for an overlap between the two types of modifiers.

ATP-Binding Cassette Transporters↗
PubMed1996

Mutation-selection balance at a modifier-of-imprinting locus.

We propose a pair of population genetic models for a modifier-of-imprinting locus for which different genotypes imprint different proportions of an imprintable target locus in their gametes. The two models examine the situations in which imprinting is advantageous, and we discuss three cases for which the modifier is respectively partially dominant, dominant, or recessive. The models predict the stable equilibrium frequencies of the mutant modifier and functionally diploid individuals in a large population in terms of up to four parameters: the mutation rate at the modifier locus, nu; the selection coefficient against the disadvantageous phenotype, sigma; the proportion of unimprinted eggs produced by homozygotes for the mutant modifier, theta, and, in the partially dominant models, the dominance parameter, kappa. The equilibrium frequency of the mutant phenotypes is shown to be approximately twice that of standard Mendelian models: 2 nu/sigma or 4nu/sigma when the modifier is recessive or dominant, respectively. Mathematical equivalences between these and nonimprinting models are noted.

Chromosome Mapping↗
PubMed1975

Efficacy of modified human immune serum globulin in the treatment of experimental murine infections with seven immunotypes of Pseudomonas aeruginosa.

Modified immune serum globulin, prepared from human immune serum globulin by a nonenzymatic method, is apparently safe for intravenous administration to humans. The efficacy of the preparation was determined in experimental murine infections with seven immunotypes of Pseudomonas aeruginosa. Intravenously administered 0.85 percent NaCl, 0.3 m glycine, and 10 percent human albumin did not protect against lethal pseudomonas infection, whereas modified immune serum globulin given by the same route did protect mice. In the mouse protection test, the mean dose of the preparation that saved 50 percent of mice infected with any of eight strains of P. aeruginosa was 480 mg/kg (range, 12-2,333 mg/kg). For five strains the 50 percent effective dose was smaller than 200 mg/kg. There was no correlation between the efficacy of modified immune serum globulin in the mouse protection test and titers of antibody, as determined by bacterial agglutination. Therapy of pseudomonas infection in mice with modified immune serum globulin was followed by a prompt and persistent decrease in the numbers of intraperitoneal bacteria. This finding is consistent with the interpretation that modified immune serum globulin acts primarily as an opsonin and not as an antitoxin. Modified immune serum globulin may prove to be useful in the treatment of human infections.

Agglutination Tests↗
PubMed2004

Reduced colonization and infection with miconazole-rifampicin modified central venous catheters: a randomized controlled clinical trial.

OBJECTIVE: Central venous catheters (CVC) are a major cause of nosocomial bloodstream infections. Catheters modified with miconazole and rifampicin that constantly and slowly release antimicrobial substances are assumed to be beneficial in reducing rates of colonization and catheter-related infections. DESIGN AND SETTING: Prospective controlled non-blinded randomized clinical trial in two German university hospitals. PATIENTS: 223 adult inpatients with CVC between October 2000 and February 2002. Baseline characteristics, APACHE II score and therapeutic interventions were comparable. INTERVENTION: Randomization to receive either a miconazole and rifampicin modified catheter (n=118) or a standard triple-lumen CVC (n=105). MEASUREMENTS, DEFINITIONS: Microbiological evaluation was done after CVC removal. A catheter was considered colonized if growth of > or =15 cfu was found by semi-quantitative roll-plate technique from a proximal or distal catheter segment. A catheter-related infection (CRI) was defined as a colonized catheter with local signs of inflammation. A catheter-related bloodstream infection (CR-BSI) was defined as a colonized catheter with isolation of the same organism from the patient's blood with accompanying clinical signs of infection. RESULTS: A colonization of CVC was observed in six patients (5.1%) with a modified catheter and 38 patients (36.2%) with a standard catheter (P < 0.001). Five patients in the modified group (4.2%) and 18 in the standard group (17.1%) developed CRI (P=0.002). One assumed CR-BSI was detected in the standard group, with none in the modified group. No adverse effects related to the modified catheters and no antimicrobial resistance were observed. CONCLUSION: CVC supersaturated with miconazole and rifampicin were associated with a significantly lower risk for catheter colonization and catheter-related infections compared to standard catheters.

Adolescent↗
PubMed2001

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR.

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.

Binding Sites↗
PubMed1981

Z-DNA conformation of N-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) determined by reactivity with anti cytidine antibodies and minimized potential energy calculations.

The conformation of poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), and calf thymus DNA modified with N-acetoxy-N-2-acetylaminofluorene (N-acetoxy-AAF) was examined by extent of reaction with anti cytidine antibodies. In contrast to modified poly(dG).poly(dC0 and DNA, modified poly(dG-dC).poly (dG-dC) failed to react with the antibodies indicating that the base pairing in this polymer is intact. This in consistent with induction of the Z-DNA conformation in AAF modified poly(dG-dC).poly(dG-dC). Using minimized potential energy calculations on the dCpdG-AAF dimer as a model for the modified polymer, it is shown that the proposed Z-DNA conformation is energetically stable. A model is proposed for an AAF modified tetramer, dGpdCpdGpdC, in which the AAF is external to the Z-DNA duplex.

2-Acetylaminofluorene↗
PubMed2004

2'-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo.

Synthesis and antisense activity of oligonucleotides modified with 2'-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2'-O-DMAOE) are described. The 2'-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide 'gapmers', with 2'-O-DMAOE- modified nucleoside residues at the ends and 2'-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. 'Gapmer' oligonucleotides have one or two 2'-O-modified regions and a 2'-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2'-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2'-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC(50) of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2'-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2'-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed.

Animals↗
PubMed2004

Thermodynamic and structural characterization of 2'-nitrogen-modified RNA duplexes.

2'-aminonucleosides are commonly used as sites of post-synthetic chemical modification within nucleic acids. As part of a larger cross-linking strategy, we appended alkyl groups onto the N2' position of 2'-amino-modified RNAs via 2'-ureido and 2'-amido linkages. We have characterized the thermodynamics of 2'-amino, 2'-alkylamido and 2'-alkylureido-modified RNA duplexes and show that 2'-ureido-modified RNAs are significantly more stable than analogous 2'-amido-modified RNAs. Using NMR spectroscopy and NMR-based molecular modeling of 2'-modified RNA duplexes, we examined the effects that 2'-nitrogen modifications have on RNA helices. Our data suggest that the 2'-ureido group forms a specific intra-nucleoside interaction that cannot occur within 2'-amido-modified helices. These results indicate that 2'-ureido modifications are superior to analogous 2'-amido ones for applications that require stable base pairing.

Magnetic Resonance Spectroscopy↗
PubMed2005

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene.

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5-8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5-100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.

Anthracenes↗
PubMed2002

Modified DNA aptamers against sweet agent aspartame.

We obtained a modified DNA aptamer against sweetener, aspartame, by in vitro selection method. The modified DNA was prepared from dATP, dGTP, dCTP and a modified dTTP bearing a terminal amino group at C-5 position in place of thymidine by PCR using a hyper thermophilic DNA polymerase, KOD Dash DNA polymerase. The synthetic 102-mer DNA with a 60-mer random region was used as an initial template for the PCR. The PCR-amplified modified DNA library was applied to an aspartame-agarose column, and then the bound modified DNA was eluted from the column for the affinity chromatography selection. Repeating the procedure, we selected the modified DNA aptamer against aspartame.

Aspartame↗
PubMed2006

Synthesis and properties of modified siRNA having amide-linked oligoribonucleosides at their 3' overhang regions.

siRNAs against luciferase gene were modified with amide-linked oligoribonucleosides (amide-linked RNA) at their 3'-overhang regions. The modified siRNAs were more stable than the unmodified siRNA against degradation by nuclease S1, which indicates that the modified overhang regions increase the nuclease resistance of the siRNAs. Tm values of the modified siRNAs increased compared with that of the unmodified siRNA. This result suggests that the modified overhang regions stabilize the duplex structure of siRNAs. The modified siRNAs reduced the target gene expression in a similar manner as the unmodified siRNA in living cells. This result indicates that the overhang modifications are tolerated for the inhibition of target gene expression.

Amides↗
PubMed1998

Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor.

It was shown that proteins modified with advanced glycation end products (AGE) are effectively endocytosed by macrophages or macrophage-derived cells in vitro, and immunohistochemical studies involving anti-AGE antibodies demonstrated the accumulation of AGE-modified proteins (AGE-proteins) in macrophage-derived foam cells in human atherosclerotic lesions in situ, suggesting the involvement of AGE-modified LDL in the atherogenic process in vivo. To examine this suggestion, LDL was modified with glycolaldehyde, a highly reactive intermediate of the Maillard reaction. Physicochemically, glycolaldehyde-modified LDL (GA-LDL) was characterized by increases in negative charge, fluorescence intensity, and reactivity to anti-AGE antibodies, properties highly similar to those of AGE-proteins. The cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL was specifically recognized and endocytosed, followed by lysosomal degradation. The endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), and the endocytic degradation of acetyl-LDL was also competed for by GA-LDL. Furthermore, incubation of GA-LDL with these macrophages and Chinese hamster ovary cells overexpressing the macrophage scavenger receptor (MSR), but not with peritoneal macrophages from MSR-knockout mice, led to the intracellular accumulation of cholesteryl esters (CE). These results raised the possibility that AGE-modified LDL, if available in situ, is taken up by macrophages mainly via MSR and then contributes to foam cell formation in early atherosclerotic lesions.

Acetaldehyde↗
PubMed1978

Characterization of phospholipase A activity of beta1-bungarotoxin from Bungarus multicinctus venom. II. Identification of the histidine residue of beta1-bungarotoxin modified by p-bromophenacyl bromide.

beta1-Bungarotoxin modified with p-bromophenacyl bromide (BPB) was reduced and carboxymethylated, and the resulting two constituent RCM-polypeptide chains (the RCM-A and B chains) were separated. The RCM-A chain was found to be modified by BPB by measuring its UV absorption spectrum and was shown to have lost one histidine residue by analyzing its amino acid composition. To determine the location of the modified histidine residue in the A chain of the toxin, the RCM-A chain was digested with TPCK-trypsin, and the resulting peptides were fractionated by gel filtration followed by DEAE-cellulose chromatography. The modified residue was finally identified as histidine-48 in the A chain by Edman degradation and from the amino acid composition of the BPB-modified peptide. The amino acid sequence around the modified histidine residue in the A chain is highly homologous with those of porcine pancreas phospholipase A2 and presynaptic toxin, notexin. We conclude that histidine-48 in the A chain participates in the phospholipase A activity of beta1-bungarotoxin.

Acetophenones↗
PubMed1984

Bindings of Ca2+ and substrate analogs to a cobra venom phospholipase A2 in which the alpha-amino group is modified to an alpha-keto group.

The pH dependence of the chemical reaction rate of p-bromophenacyl bromide (BPB) with His 48 of cobra (Naja naja atra) venom phospholipase A2, in which the alpha-NH2 group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 in the absence of Ca2+. The pH-dependence curve was monophasic with a midpoint at pH 7.9, which corresponds to the pK value of His 48 of the alpha-NH2-modified enzyme, whereas the curve for the intact enzyme was biphasic, indicating participation of two ionizable groups with pK values of 7.3 and 8.55 (Teshima et al. (1982) J. Biochem. 91, 1778-1788). These two groups were thus identified as His 48 and the alpha-NH2 group, respectively. The pH dependence of the binding constant of Ca2+ to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by measuring the tryptophyl fluorescence changes. The pH-dependence curve was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 13-20), and it was interpreted in terms of participation of His 48 and Asp 49 (pK 5.4). The absence of participation of the alpha-NH2 group in the Ca2+ binding was thus confirmed. Bindings of monodispersed n-dodecylphosphorylcholine (n-C12PC) and micellar n-hexadecylphosphorylcholine (n-C16PC) to the alpha-NH2-modified enzyme were studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) and tryptophyl fluorescence methods, respectively. The binding constant of the monodispersed substrate was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The binding constant of the micellar substrate to the modified enzyme in the presence of Ca2+ was also very similar to that for the intact enzyme-Ca2+ complex (Teshima et al. (1983) J. Biochem. 94, 223-232), and the pH-dependence curve was interpreted in terms of participation of His 48. On the other hand, the binding constant of the micellar substrate to the modified apoenzyme was much smaller than that for the intact apoenzyme. Nevertheless, the pH-dependence curve could be interpreted in terms of participation of His 48 and Asp 49. From these findings, it was concluded that the ionization state of the alpha-NH2 group of cobra venom phospholipase A2 is essentially irrelevant to the bindings of Ca2+ and also of the monodispersed and micellar substrates.

Animals↗