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  • Article
    Wallace JE.
    Br Med J. 1937 Jun 05;1(3987):1151-1168.3.
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  • Article
    Grandchamp B, Bissell DM, Licko V, Schmid R.
    J Biol Chem. 1981 Nov 25;256(22):11677-83.
    Studies with the intact liver have suggested that newly synthesized heme exists transiently in a small pool before its incorporation into tissue heme proteins. The same or a closely related pool may regulate synthesis of heme and serve as the precursor of "early peak" bilirubin. To delineate this postulated pool by a direct approach, we have utilized primary cultures of adult rat hepatocytes. Cultures pulse-labeled with delta-amino[3H]levulinic acid at various time points were fractionated into 105,000 X g supernatant and pellet. Labeled heme appeared within 1 to 2 min in the cytosol fraction, followed by transfer to the pellet. The kinetics of heme formation and transfer and of labeled bilirubin production were analyzed by computer simulation utilizing the least squares method. The experimental findings conformed best to a four-compartment model that includes a second cytosolic heme compartment exchanging with the initially labeled compartment but not serving as a direct precursor of bilirubin. Calculation of apparent rate coefficients indicated that, in cultured hepatocytes, 20% of newly formed heme is converted directly to bile pigment, whereas 80% is utilized for formation of cellular heme proteins (64% in the pellet, 16% in the second cytosol compartment). This experimental approach has provided direct evidence for a rapidly formed cytosolic heme fraction which appears to be identical with the previously postulated regulatory or "unassigned" heme pool of the liver.
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  • Article
    Wu LN, Fisher RR.
    J Biol Chem. 1982 Oct 10;257(19):11680-3.
    Bovine heart submitochondrial particle energy-linked NADH leads to NADP+ transhydrogenase also catalyzes transhydrogenation between NADPH and NADP+. The stereochemistry of hydride ion transfer in the NADH leads to NADP+ reaction involves the nicotinamide 4A locus of NADH and the 4B locus of NADPH. In this paper it is demonstrated that during NADPH leads to NADP+ transhydrogenation the NADP+ is reduced exclusively at the 4B locus and that oxidation of NADPH is predominately at the 4B locus. Reduction of [4-3H]NADP+ by NADPH yielded [4A-3H]NADPH as the only product. Oxidation of [4A-3H]NADPH by NADP+ resulted in the conversion of nearly 33% of the label into [4-3H]NADP+, whereas the oxidation of [4B-3H] NADPH yielded only about a 6.5% conversion. These data suggest that while a small portion of total energy-linked NADPH leads to NADP+ transhydrogenation results from the binding of NADPH at the NAD domain of the active site with hydride transfer to NADP+ bound at the NADP domain, most of the reaction occurs by a mechanism in which both substrates are bound sequentially at the NADP domain. It is proposed that NADPH leads to NADP+ transhydrogenation represents a partial reaction of NADH leads to NADP+ transhydrogenation which involves the participation of a reduced-enzyme intermediate.
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  • Article
    Muszynski M, Birnbaum RS, Roos BA.
    J Biol Chem. 1983 Oct 10;258(19):11678-83.
    A rat medullary thyroid carcinoma cell line, CA-77, has been established as a model system for investigating calcitonin biosynthesis and secretion. Growth of this cell line in serum-free defined medium provided suitable conditions for studying steroid hormone effects on the production of calcitonin and related peptides. After exposure for 5 days to a variety of steroids, only dexamethasone and corticosterone increased cellular content of calcitonin and a second secretory peptide (CCAP) derived from the same mRNA translation product as calcitonin. Glucocorticoids had no effect on cellular somatostatin, another secretory product of these cells. Increasing doses of dexamethasone progressively elevated cellular calcitonin and CCAP, with a maximal effect at 10(-8) M; 10(-9) M and lower doses were ineffective. On a molar basis, corticosterone was approximately 50-fold less potent than the synthetic glucocorticoid. An increase in cellular calcitonin content was observed only after 48 h of glucocorticoid treatment; a maximum increase (13-fold) occurred after 7 days. Glucocorticoids also increased basal calcitonin secretion. Similar effects were observed for cellular and secreted CCAP. Withdrawal of dexamethasone after 4 days of treatment lowered cellular calcitonin toward the level of control cultures. Dexamethasone pretreatment potentiated the acute secretory response to calcium for both calcitonin and CCAP, while no such enhancement was noted for calcium stimulation of somatostatin secretion. We conclude that the glucocorticoids specifically stimulate the production and secretion of calcitonin and CCAP, two secretory peptides derived from preprocalcitonin.
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  • Article
    Bullough DA, Verburg JG, Yoshida M, Allison WS.
    J Biol Chem. 1987 Aug 25;262(24):11675-83.
    The characteristics of ATP hydrolysis at a single catalytic site of the bovine heart F1-ATPase (MF1) as originally described by Grubmeyer et al. (Grubmeyer, C., Cross, R.L., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100) were compared with those of various chemically modified preparations of MF1 in which the steady state activity was severely attenuated. Although it was not necessary to age our preparations of native MF1 in the presence of 2 mM Pi to observe the same characteristics of single site catalysis, such aging did shift the equilibrium of bound substrate and bound products at the single catalytic site in favor of ATP. After loading a single catalytic site on the enzyme with substoichiometric [alpha,gamma-32P]ATP, the addition of 5-20 microM ATP or ADP was effective in promoting both the hydrolysis of bound [alpha,gamma-32P]ATP and release of radioactive products. Under these conditions, the 5-20 microM ATP added as promoter was hydrolyzed at a rate commensurate with the turnover rate of the enzyme, whereas the promoted hydrolysis of the [alpha,gamma-32P]ATP, preloaded at a single catalytic site, was considerably slower. Therefore, the high affinity, single catalytic site loaded first does not directly contribute to steady state ATP hydrolysis. That the single, high affinity catalytic site is not a "normal" catalytic site is supported by the properties of enzyme modified by 5'-p-fluorosulfonylbenzoyladenosine which exhibits only slightly altered characteristics of single site catalysis and promoted single site catalysis, despite exhibiting severely attenuated steady state turnover. Other modified forms of the enzyme in which the steady state activity was severely attenuated by derivatization with 5'-p-fluorosulfonylbenzoylinosine, 7-chloro-4-nitrobenzofurazan, or 1,5-difluoro-2,4-dinitrobenzene also bound substoichiometric ATP at a single catalytic site. However, the characteristics of single site hydrolysis by these modified forms of the enzyme differed considerably from those of native MF1.
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  • Article
    Vlahos CJ, Dekker EE.
    J Biol Chem. 1988 Aug 25;263(24):11683-91.
    The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.
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  • Article
    Hickmott TW.
    Phys Rev B Condens Matter. 1989 Dec 15;40(17):11683-11692.
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  • Article
    Mandal P, Tiwari RS, Srivastava ON.
    Phys Rev B Condens Matter. 1991 Dec 01;44(21):11683-11688.
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  • Article
    Schneegurt MA, Beale SI.
    Biochemistry. 1992 Dec 01;31(47):11677-83.
    Chlorophyll (Chl) b is an accessory light-harvesting pigment of plants and chlorophyte algae. Chl b differs from Chl a in that the 3-methyl group on ring B of chl a is replaced by a 3-formyl group on Chl b. The present study determined the biosynthetic origin of the Chl b formyl oxygen in in vivo labeling experiments. A mutant strain of the unicellular chlorophyte Chlorella vulgaris, which can not synthesize Chls when cultured in the dark but rapidly greens when transferred to the light, was grown in the dark for several generations to deplete Chls, and then the cells were transferred to the light and allowed to form Chls in a controlled atmosphere containing 18O2. Chl a and Chl b were purified from the cells and analyzed by high-resolution mass spectroscopy. Analysis of the mass spectra indicated that over 76% of the Chl a molecules had incorporated an atom of 18O. For Chl b, 58% of the molecules had incorporated an atom of 18O at one position and 34% of the molecules had incorporated an atom of 18O at a second position. These results demonstrate that the isocyclic ring keto oxygen of both Chl a and Chl b, as well as the formyl oxygen of Chl b, is derived from O2.
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  • Article
    Sureau A, Soret J, Vellard M, Crochet J, Perbal B.
    Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11683-7.
    We have previously reported that expression of the c-myb gene in normal avian thymic cells proceeds through the intermolecular recombination of ET (thymus-specific) and c-myb coding sequences, thereby generating a novel type of c-myb product. Antisense transcripts expressed from the ET locus encode the extremely well-conserved splicing factor PR264/SC35. We now show that the human PR264 promoter sequences contain several myb-recognition elements that efficiently interact in vitro with the c-myb DNA-binding domain. Moreover, expression from the PR264 promoter is transactivated, both in vitro and in cultured cells, by different c-myb products. Thus, the PR264 gene is most likely a physiological target for the c-myb family of transcription factors.
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  • Article
    Lanza P, Billetta R, Antonenko S, Zanetti M.
    Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11683-7.
    Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.
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  • Article
    Bogazzi F, Hudson LD, Nikodem VM.
    J Biol Chem. 1994 Apr 22;269(16):11683-6.
    Retinoid-like receptors play a central role in hormonal responses by forming heterodimers with other nuclear hormone receptors. In this study we have identified the peroxisome proliferator-activated receptor (PPAR) as a new thyroid hormone receptor (THR) auxiliary nuclear protein, heterodimerizing with THR in solution. Although these heterodimers do not recognize a classical thyroid hormone response element (TRE) characterized by direct repeat separated by four nucleotides (DR+4), PPAR behaves as a dominant negative regulator of thyroid hormone (TH) action. However, a TH-dependent positive effect is elicited by selective interaction of the THR beta-PPAR but not the THR alpha-PPAR heterodimer with a novel TRE (DR+2). The critical region of THR beta was mapped to 3 amino acids in the distal box of the DNA binding domain. Hence, PPAR can positively or negatively influence TH action depending on TRE structure and THR isotype.
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  • Article
    Yamada T, Uyeda A, Kidera A, Kikuchi M.
    Biochemistry. 1994 Oct 04;33(39):11678-83.
    To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)
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  • Article
    Abounadi A, Di Blasio M, Bouchara D, Calas J, Averous M, Briot O, Briot N, Cloitre T, Aulombard RL, Gil B.
    Phys Rev B Condens Matter. 1994 Oct 15;50(16):11677-11683.
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  • Article
    Sugimoto M, Oohashi T, Ninomiya Y.
    Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11679-83.
    The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues.
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  • Article
    Eder R, Ohta Y.
    Phys Rev B Condens Matter. 1995 May 01;51(17):11683-11689.
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  • Article
    Wojcikiewicz RJ.
    J Biol Chem. 1995 May 12;270(19):11678-83.
    The type I inositol 1,4,5-trisphosphate (InsP3) receptor can be rapidly depleted from cells during stimulation of phosphoinositide hydrolysis because its degradation is accelerated (Wojcikiewicz, R. J. H., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994) J. Biol. Chem. 269, 7963-7969). The present study examines the regulatory properties of type II and III InsP3 receptors. Initially, the relative abundance of InsP3 receptors was defined in a range of cell types by quantitative immunoblotting. These studies showed that the proportions in which type I, II, and III InsP3 receptors are expressed differs greatly and that some cells (for example, AR4-2J rat pancreatoma cells) express all three receptors. Analysis of the effects of cholecystokinin and bombesin on AR4-2J cells showed that each of the InsP3 receptors could be down-regulated during activation of phosphoinositide hydrolysis, but that depletion of the type II receptor was limited. Such a discrepancy was also seen in rat cerebellar granule cells and was found to result from the type II receptor being relatively resistant to degradation. In conclusion, type I, II, and III receptors can all be down-regulated, but with different characteristics. As the relative abundance of InsP3 receptors is extremely variable, the extent to which activation of the down-regulatory process alters intracellular signaling will vary depending on which InsP3 receptors are expressed.
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  • Article
    Communi D, Lecocq R, Erneux C.
    J Biol Chem. 1996 May 17;271(20):11676-83.
    The crucial role of two reactive arginyl residues within the substrate binding domain of human Type I D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 5-phosphatase has been investigated by chemical modification and site-directed mutagenesis. Chemical modification of the enzyme by phenylglyoxal is accompanied by irreversible inhibition of enzymic activity. Our studies demonstrate that phenylglyoxal forms an enzyme-inhibitor complex and that the modification reaction is prevented in the presence of either Ins(1,4,5)P3, D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) or 2,3-bisphosphoglycerate (2,3-BPG). Direct [3H]Ins(1,4,5)P3 binding to the covalently modified enzyme is dramatically reduced. The stoichiometry of labeling with 14C-labeled phenylglyoxal is shown to be 2.1 mol of phenylglyoxal incorporated per mol of enzyme. A single [14C]phenylglyoxal-modified peptide is isolated following alpha-chymotrypsin proteolysis of the radiolabeled Ins(1,4,5)P3 5-phosphatase and reverse-phase high performance liquid chromatography (HPLC). The peptide sequence (i.e. M-N-T-R-C-P-A-W-C-D-R-I-L) corresponds to amino acids 340-352 of Ins(1,4,5)P3 5-phosphatase. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivatives shows the modified amino acids to be Arg-343 and Arg-350. Furthermore, two mutant enzymes were obtained by site-directed mutagenesis of the two arginyl residues to alanine, and both mutant enzymes have identical UV circular dichroism (CD) spectra. The two mutants (i.e. R343A and R350A) show increased Km values for Ins(l,4,5)P3 (10- and 15-fold, respectively) resulting in a dramatic loss in enzymic activity. In conclusion, we have directly identified two reactive arginyl residues as part of the active site of Ins(1,4,5)P3 5-phosphatase. These results point out the crucial role for substrate recognition of a 10 amino acids-long sequence segment which is conserved among the primary structure of inositol and phosphatidylinositol polyphosphate 5-phosphatases.
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  • Article
    Spudich G, Marqusee S.
    Biochemistry. 2000 Sep 26;39(38):11677-83.
    Experimental studies of protein stability often rely on the determination of an "m value", which describes the denaturant dependence of the free energy change between two states (DeltaG = DeltaG(H2O) - m[denaturant]). Changes in the m value accompanying site specific mutations are usually attributed to structural alterations in the native or unfolded ensemble. Here, we provide an example of significant reduction in the m value resulting from a subtle deviation in two-state behavior not detected by traditional methods. The protein that is studied is a variant of Escherchia coli RNase H in which three residues predicted to be involved in a partially buried salt bridge network were mutated to alanine (R46A, D102A, and D148A). Equilibrium denaturant profiles monitored by both fluorescence and circular dichroism appeared to be cooperative, and a two-state analysis yielded a DeltaG(UN) of approximately -3 kcal/mol with an m value of 1.4 kcal mol(-1) M(-1) (vs 2.3 for RNase H). Analysis of kinetic refolding experiments suggests that the system is actually three-state at equilibrium with an appreciable concentration of an intermediate state under low denaturant concentrations. The stability of the native state determined from a fit of these kinetic data is -6.7 kcal/mol, suggesting that the stability determined by traditional two-state equilibrium analysis is a gross underestimate. The only hint to this loss of two-state behavior was a decrease in the apparent m value, and the presence of the equilibrium intermediate was only identified by a kinetic analysis. Our work serves as a cautionary note; the possibility of a three-state system should be closely addressed before interpreting a change in the m value as a change in the native or unfolded state.
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  • Article
    Kass RS, Cabo C.
    Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11683-4.
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