DOCSLIB.ORG

Insight Into Structural Aspects of Histidine 284 of Daphnia Magna

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Insight Into Structural Aspects of Histidine 284 of Daphnia Magna Molecules and Cells Insight into Structural Aspects of Histidine 284 of Daphnia magna Arginine Kinase Zhili Rao1,4, So Young Kim1,4, Xiaotong Li1, Da Som Kim1, Yong Ju Kim2,3,*, and Jung Hee Park1,3,* 1Division of Biotechnology, College of Environmental & Bioresources Sciences, Jeonbuk National University, Iksan 54596, Korea, 2Department of Herbal Medicine Resources, College of Environmental and Bioresource Sciences, Jeonbuk National University, Iksan 54596, Korea, 3Advanced Institute of Environment and Bioscience, College of Environmental & Bioresources Sciences, Jeonbuk National University, Iksan 54596, Korea, 4These authors contributed equally to this work. *Correspondence: [email protected] (YJK); [email protected] (JHP) https://doi.org/10.14348/molcells.2020.0136 www.molcells.org Arginine kinase (AK), a bioenergy-related enzyme, is INTRODUCTION distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, Phosphagen kinases (PKs) are a family of phosphotransfer- the highly conserved histidine 284 (H284) in AK of Daphnia ases that reversibly catalyze the delivery of high-energy phos- magna (DmAK) was replaced with alanine to elucidate the phoryl groups between ATP and guanidine derivatives. The role of H284. We examined the alteration of catalytic activity phosphorylated guanidines, also known as phosphagens, are and structural changes of H284A in DmAK. The catalytic a type of high-energy compounds that play an important role activity of H284A was reduced dramatically compared to in cellular energy homeostasis in both the muscles and the that in wild type (WT). Thus the crystal structure of H284A brain by providing a way to store high-energy phosphates displayed several structural changes, including the alteration as a metastable compound (Ellington, 2001). PKs are wide- of D324, a hydrogen-bonding network around H284, and spread in all types of animal cells that require considerable the disruption of π-stacking between the imidazole group of amounts of energy. Eight PKs have been reported in inverte- the H284 residue and the adenine ring of ATP. These findings brates (Ellington, 1989; Uda et al., 2005). One of these, ar- suggest that such alterations might affect a conformational ginine kinase (AK), is distributed widely in invertebrates, and change of the specific loop consisting of G310-V322 at its activity has also been observed in arthropods, mollusks, the antiparallel β-sheet region. Thus, we speculated that nematodes, protozoans, and some bacteria (Ellington, 1989; the H284 residue might play an important role in the Newsholme et al., 1978). conformational change of the specific loop when ATP binds Arginine, which is an essential amino acid in many or- to the substrate-binding site of DmAK. ganisms, is produced as an intermediate in two regulatory cycles: the urea and the nitric oxide cycles (Noh et al., 2002). Keywords: arginine kinase, crystallization, kinetics, point This intermediate is also used by AK to maintain energy mutation, X-ray crystallography homeostasis in cells. AK requires both arginine and ATP as bi-substrates and plays a key role in catalyzing the reversible transfer of the γ-phosphoryl group of ATP to the guanidine functional group of arginine (Adeyemi and Whiteley, 2014; Alonso et al., 2001; Hansen and Knowles, 1981; Wyss and Received 19 June, 2020; revised 29 July, 2020; accepted 10 August, 2020; published online 31 August, 2020 eISSN: 0219-1032 ©The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. 784 Mol. Cells 2020; 43(9): 784-792 The Role of H284 in Structural Change of the Specific Loop Zhili Rao et al. Kaddurah-Daouk, 2000; Zhou et al., 1998). In particular, tion of H284A to DmAK, some structural alterations of the AK activity is required for muscle contraction and the gen- surrounding residue were detected, i.e., the disruption of eration of electrical conductance in nerves. Many functional π-stacking between the imidazole group of the H284 residue and structural studies of AKs from diverse organisms have and the adenine ring of ATP, the alteration of D324, and a been reported in a substrate-binding-dependent manner hydrogen-bonding network around H284. These findings (Azzi et al., 2004; Brown and Grossman, 2004; Fernandez may be important clues for explaining the effects of H284A et al., 2007; Niu et al., 2011; Pruett et al., 2003; Watts et on the reduction of activity compared with WT. Therefore, al., 1980). The overall topology of AKs commonly has two our results provide a clue to improve our understanding of different domains: an N-terminal domain, which consists of the bioenergetics mechanism. small α-helices, and a C-terminal domain, which comprises an eight-strand antiparallel β-sheet connected by α-helices. In MATERIALS AND METHODS AKs, the N-terminal domain can interact with arginine (i.e., either L-arginine or phosphoarginine), whereas the central Cloning, expression, and purification of DmAK WT and role of the C-terminal domain is nucleotide-binding (i.e., ei- H284A ther ATP or ADP) (Fernandez et al., 2007; Zhou et al., 1998). The DmAK gene was synthesized according to the full-length In the structural comparison between Trypanosoma cruzi open reading frame (GenBank accession No. AID69955.1) AKapo (Tc AKapo) and Limulus Polyphemus AKholo (LpAKholo), from National Center for Biotechnology Information (NCBI; some structural changes accompanied by rigid-body motion http://www.ncbi.nlm.nih.gov). The synthesized gene was of the helix and the antiparallel β-sheet outward from the nu- amplified by a standard PCR method with a primer set (5′- cleotide-binding site in the C-terminal domain were observed GCACTCCATATGCATCACCATCATCATCATGTGGAC-3′ and 5′- significantly (Clark et al., 2012; Fernandez et al., 2007). GCACTCGCGG CCGCTTATGCGGC TTC-3′) and inserted into In addition, the structural changes in the substrate-bound an expression vector (pET30a; New England Biolabs, USA) (holo) and the substrate-free (apo) state of AK have been using NdeI and NotI restriction enzyme sites. A six-His tag was confirmed by joint crystallographic and NMR residual dipolar fused to the N-terminus of the gene to facilitate protein pu- coupling analysis (Niu et al., 2011). Thus, the results indicated rification. The mutation was introduced by modified overlap that the two different AK conformations might derive from PCR using a single site mismatched primer against H284 (5′- substrate-induced motion. GCAGTGCAATGGCCACTGAGGCCC-3′). The recombinant Several highly conserved residues in the arginine and ATP construct was transformed into Escherichia coli BL21 (DE3) binding sites that alter AK enzyme activity have been report- cells. Then, overnight cultures of the transformed cells were ed (Guo et al., 2004; Strong and Ellington, 1996; Takeuchi et transferred to 1 L of LB broth (BD Bioscience, USA) contain- al., 2004; Wu et al., 2014). In Nautilus and Stichophus AKs, ing kanamycin (Duchefa Biochemie, The Netherlands), with the mutations of D62G and R193G accompanying the disap- a final concentration of 50 μg/ml, and incubated at 37°C pearance of the salt bridge showed considerably decreased and 200 rpm until reaching the optical density of 0.6 at 600 activity. Hence, the mutagenesis of both D62G and R193G nm (OD600). Large quantities of recombinant DmAK proteins has been proposed to be associated with structural stability were obtained by inducing the expression of DmAK WT and and bi-substrates’ affinity (Suzuki et al., 2000a; 2000b). On H284A with 0.5 mM isopropyl β-D-1-thiogalactopyranoside the other hand, the mutations of C271, P272 and T273 (IPTG) (Duchefa Biochemie) for 4 h at 18°C and 170 rpm. replaced with alanine, which are located at the hinge loop The harvested cells were resuspended in 20 ml of buffer (10 in AKs, showed reduced activity in the previous studies (Liu mM Tris-HCl, pH 8.0, 100 mM NaCl) and lysed for 2 min by et al., 2011; Strong and Ellington, 1996; Wu et al., 2008; sonication (4 s sonication and 4 s rest) (Sonics and Materi- 2014). These findings revealed an alteration of enzyme activi- als, USA). The cellular debris was removed by centrifugation ty due to structural changes at the arginine binding site. Thus, (12,000 rpm, 4°C) for 20 min, and then the supernatant was these residues were confirmed as key players in the substrate purified using affinity chromatography with a HisTrap FF col- synergism between the arginine residues (L-arginine and umn (GE Healthcare, USA) (Rao et al., 2019). To ensure the phosphoarginine) of AK and their constraining position (Wu high purity of DmAK WT and H284A, the HiTrap Q FF column et al., 2014). In the transition state analog (TSA) including (GE Healthcare) was used with a double gradient elution nitrate (NO3), the crystal structure of AK from horseshoe crab buffer system (10 mM Tris-HCl, pH 7.0, 1 M NaCl). The puri- revealed the relationship between nitrate and the bi-sub- fied DmAK was analyzed by 12% SDS-PAGE (Supplementary strate (Zhou et al., 1998). The substrate arginine showed Method), and Bradford’s method was employed to ascertain two salt bridges with E225 and E314. Hence, the variants of the protein concentration (Bradford, 1976). E225 and E314 exhibited alteration of catalytic activity with a reduced rate of production of phosphoarginine and ADP Enzyme assay and determination of kinetic parameters (Pruett et al., 2003). To ascertain the optimal pH and temperature, AK activity was Although several mutagenesis studies have reported highly conducted as described previously (Li et al., 2006; Pereira et conserved residues at the arginine binding site of AKs, a role al., 2000; Wu et al., 2014).
Load more

Recommended publications
  • (12) Patent Application Publication (10) Pub. No.: US 2010/0317005 A1 Hardin Et Al
    US 20100317005A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0317005 A1 Hardin et al. (43) Pub. Date: Dec. 16, 2010 (54) MODIFIED NUCLEOTIDES AND METHODS (22) Filed: Mar. 15, 2010 FOR MAKING AND USE SAME Related U.S. Application Data (63) Continuation of application No. 11/007,794, filed on Dec. 8, 2004, now abandoned, which is a continuation (75) Inventors: Susan H. Hardin, College Station, in-part of application No. 09/901,782, filed on Jul. 9, TX (US); Hongyi Wang, Pearland, 2001. TX (US); Brent A. Mulder, (60) Provisional application No. 60/527,909, filed on Dec. Sugarland, TX (US); Nathan K. 8, 2003, provisional application No. 60/216,594, filed Agnew, Richmond, TX (US); on Jul. 7, 2000. Tommie L. Lincecum, JR., Publication Classification Houston, TX (US) (51) Int. Cl. CI2O I/68 (2006.01) Correspondence Address: (52) U.S. Cl. ............................................................ 435/6 LIFE TECHNOLOGES CORPORATION (57) ABSTRACT CFO INTELLEVATE Labeled nucleotide triphosphates are disclosed having a label P.O. BOX S2OSO bonded to the gamma phosphate of the nucleotide triphos MINNEAPOLIS, MN 55402 (US) phate. Methods for using the gamma phosphate labeled nucleotide are also disclosed where the gamma phosphate labeled nucleotide are used to attach the labeled gamma phos (73) Assignees: LIFE TECHNOLOGIES phate in a catalyzed (enzyme or man-made catalyst) reaction to a target biomolecule or to exchange a phosphate on a target CORPORATION, Carlsbad, CA biomolecule with a labeled gamme phosphate. Preferred tar (US); VISIGEN get biomolecules are DNAs, RNAs, DNA/RNAs, PNA, BIOTECHNOLOGIES, INC. polypeptide (e.g., proteins enzymes, protein, assemblages, etc.), Sugars and polysaccharides or mixed biomolecules hav ing two or more of DNAs, RNAs, DNA/RNAs, polypeptide, (21) Appl.
    [Show full text]
  • Identi Cation of Metabolic Reprogramming Related Gene
    Identication of Metabolic Reprogramming Related Gene Signature to Predict the Prognosis of Bladder Cancer Patients Tinghao Li The First Aliated Hospital of Chongqing Medical University https://orcid.org/0000-0002-7980-6298 Hang Tong The First Aliated Hospital of Chongqing Medical University Hubin Yin The First Aliated Hospital of Chongqing Medical University Honghao Cao Rongchang tranditional Chinese medicine hospital Junlong Zhu the rst aiated hospital of Chongqing medical uiversity Zijia Qin The First Aliated Hospital of Chongqing Medical University Siwen Yin The First Aliated Hospital of Chongqing Medical University Weiyang He ( [email protected] ) The First Aliated Hospital of Chongqing Medical University https://orcid.org/0000-0002-7445-8781 Research article Keywords: Metabolic reprogramming, Prognosis, Bladder cancer, TCGA, GEO Posted Date: December 23rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-132465/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract Background: Different kinds of metabolic reprogramming have been widely researched in multifarious cancer types and show up as a guaranteed prognostic predictor, while bladder cancer (BLCA) is most frequent urothelium carcinoma but with poor prognosis despite there are emerging treatments, for lack of reliable predicting biomarkers to early predict the prognosis and delayed treatment options for patients in the terminal stage. Our study aims to explore new prognostic factors related to metabolism in BLCA and make these genes up as novel risk stratication. Methods: We selected a large number of samples downloaded from TCGA (The Cancer Genome Atlas) to nd out the possible glycolysis-related genes that correlated with differentiation from cancer sample to normal tissue, aimed to nd out a more credible model.
    [Show full text]
  • Some Catalytic and Regulatory Properties of Pyruvate Kinase from the Spadix and Retractor Muscles of Nautilus Pompilius1
    Pacific Science (1982), vol. 36, no. 3 © 1983 by the University of Hawaii Press. All rights reserved Some Catalytic and Regulatory Properties of Pyruvate Kinase from the Spadix and Retractor Muscles of Nautilus pompilius1 JEREMY H. A. FIELDS 2 ABSTRACT: Pyruvate kinase was partially purified from the spadix and retrac­ tor muscles of Nautilus pompilius. In both cases, the enzyme was activated by magnesium and potassium ions with similar affinities (apparent Ka values were 0.63 ± 0.04 mM and 5.8 ± 0.4 mM, respectively, for the enzyme from the spadix; and 0.77 ± 0.06 mM and 6.7 ± 0.8 mM, respectively, for the enzyme from the retractor muscle). The enzymes showed normal hyperbolic saturation kinetics for the substrates adenosine Y-diphosphate and phosphoenolpyruvate, and the apparent Km values were identical when measured at saturating concen­ trations of the cosubstrate (apparent Km values were 0.28 ± 0.01 mM and 0.063 ± 0.005 mM, respectively, for the spadix). Adenosine 5'-triphosphate, alanine, and citrate were found to be inhibitors. The enzyme from the spadix was more susceptible to inhibition by alanine than that from the retractor muscle. For the latter enzyme, inhibition by alanine was noncompetitive with respect to phosphoenolpyruvate, but the inhibition was nonlinear; it also decreased the affinity for Mg2+. For the enzyme from the spadix, inhibition by alanine changed the saturation kinetics for phosphoenolpyruvate to sigmoidal form. The affinity for Mg2+ was also decreased by alanine. For both enzymes, fructose-I, 6-bisphosphate at a concentration of 0.05 mM partially reversed the inhibition by alanine, but not that by adenosine Y-triphosphate.
    [Show full text]
  • S42003-020-01354-W.Pdf
    ARTICLE https://doi.org/10.1038/s42003-020-01354-w OPEN Broad-complex transcription factor mediates opposing hormonal regulation of two phylogenetically distant arginine kinase genes in Tribolium castaneum 1234567890():,; Nan Zhang1, Heng Jiang1, Xiangkun Meng1, Kun Qian1, Yaping Liu1, Qisheng Song 2, David Stanley3, Jincai Wu1, ✉ Yoonseong Park4 & Jianjun Wang1 The phosphoarginine-arginine kinase shuttle system plays a critical role in maintaining insect cellular energy homeostasis. Insect molting and metamorphosis are coordinated by fluc- tuations of the ecdysteroid and juvenile hormone. However, the hormonal regulation of insect arginine kinases remain largely elusive. In this report, we comparatively characterized two arginine kinase genes, TcAK1 and TcAK2,inTribolium castaneum. Functional analysis using RNAi showed that TcAK1 and TcAK2 play similar roles in adult fertility and stress response. TcAK1 was detected in cytoplasm including mitochondria, whereas TcAK2 was detected in cytoplasm excluding mitochondria. Interestingly, TcAK1 expression was negatively regulated by 20-hydroxyecdysone and positively by juvenile hormone, whereas TcAK2 was regulated by the opposite pattern. RNAi, dual-luciferase reporter assays and electrophoretic mobility shift assay further revealed that the opposite hormonal regulation of TcAK1 and TcAK2 was mediated by transcription factor Broad-Complex. Finally, relatively stable AK activities were observed during larval-pupal metamorphosis, which was generally consistent with the con- stant ATP levels. These results provide new insights into the mechanisms underlying the ATP homeostasis in insects by revealing opposite hormonal regulation of two phylogenetically distant arginine kinase genes. 1 College of Horticulture and Plant Protection, Yangzhou University, 225009 Yangzhou, China. 2 Division of Plant Sciences, University of Missouri, Columbia, MO, USA.
    [Show full text]
  • Design and Evaluation of Hmp Kinase Analogs As Thiamine
    DESIGN AND EVALUATION OF HMP KINASE ANALOGS AS THIAMINE PATHWAY INHIBITORS by DIEGO A. LOPEZ Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON Summer 2016 Copyright © by Diego A. Lopez 2016 All Rights Reserved ii Acknowledgements This work would not have been possible without the help, support, and motivation of many people. First and foremost, I would like to thank Dr. Frank W. Foss Jr. for his mentorship, guidance, and amazing contribution into my life as a young scientist. I will always look up to him with admiration and follow his academic lessons and life teachings wherever I go. I would like to express my sincere gratitude to my committee members, Dr. Jeon and Dr. Mandal for their continuous support throughout the years and for their insightful suggestions towards the development of the project. In addition, I would like to thank the Organic division professors, Dr. Lovely, Dr. Bugarin, and Dr. Pierce for always being cordial and supportive both inside and outside of the chemistry field. I would also like to thank the numerous people who have contributed into the project, specially Dr. Sumit Bhawal who started the HMP project and developed most of the syntheses present in this study. His hard-work, passion for chemistry, and strive for success will always be remembered by this colleague and friend. I thank all my undergraduate students and members of the Foss’ lab for all the hard work, merit, and joy that have brought into my academic career: Aaron, Andra, Mohammad “Mu”, Pawan, Shakar, Johny, Twi, Nicky, Kevin and Caitlynn.
    [Show full text]
  • New Quinoxaline 1,4-Di-N-Oxide Derivatives
    New quinoxaline 1,4-di-N-oxide derivatives: Trypanosomaticidal activities and enzyme docking simulation Yannick Estevez1, Miguel Quiliano2, Asuncion Burguete3, Mirko Zimic2, Edith Málaga4, Manuela Verástegui4 , Silvia Pérez-Silanes3, Ignacio Aldana3, Antonio Monge3, Denis Castillo2, Eric Deharo1,5 1. Université de Toulouse ; UPS ; UMR 152 (Laboratoire de pharmacochimie des substances naturelles et pharmacophores redox), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France. 2. Bioinformatics Unit - Drug Design Group. Laboratorios de Investigación y Desarrollo. Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima, Perú. 3. Unidad de Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, Campus Universitario, Pamplona, Spain. 4. Laboratorio de Enfermedades Infecciosas. Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia (UPCH), Av. Honorio Delgado 430, SMP, Lima, Peru. 5. IRD ; UMR-152 ; Mission IRD Casilla 18-1209 Lima, Peru. These authors contributed equally to this work and should be considered co-first authors. 1 RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) CORRESPONDING AUTHOR FOOTNOTE to whom correspondence should be addressed: phone (+ 51) 1-441-32-23. E-mail : [email protected] Abstract Two series of pyrazol and propenone quinoxaline derivatives were tested for parasiticidal activity (against amastigotes of Leishmania peruviana and trypomastigotes of Trypanosoma cruzi) and for toxicity against proliferative and non-proliferative cells. The pyrazol series was almost inactive against T. cruzi but, 2,6-Dimethyl-3-[5-(3,4,5-trimethoxy-phenyl)-4,5-dihydro-1H-pyrazol-3-yl] - quinoxaline 1,4-dioxide inhibited 50% of Leishmania growth at 8.9 µM with no impact against proliferative kidney cells and low toxicity against Thp-1 and murine macrophages.
    [Show full text]
  • Supplemental Table S1: Comparison of the Deleted Genes in the Genome-Reduced Strains
    Supplemental Table S1: Comparison of the deleted genes in the genome-reduced strains Legend 1 Locus tag according to the reference genome sequence of B. subtilis 168 (NC_000964) Genes highlighted in blue have been deleted from the respective strains Genes highlighted in green have been inserted into the indicated strain, they are present in all following strains Regions highlighted in red could not be deleted as a unit Regions highlighted in orange were not deleted in the genome-reduced strains since their deletion resulted in severe growth defects Gene BSU_number 1 Function ∆6 IIG-Bs27-47-24 PG10 PS38 dnaA BSU00010 replication initiation protein dnaN BSU00020 DNA polymerase III (beta subunit), beta clamp yaaA BSU00030 unknown recF BSU00040 repair, recombination remB BSU00050 involved in the activation of biofilm matrix biosynthetic operons gyrB BSU00060 DNA-Gyrase (subunit B) gyrA BSU00070 DNA-Gyrase (subunit A) rrnO-16S- trnO-Ala- trnO-Ile- rrnO-23S- rrnO-5S yaaC BSU00080 unknown guaB BSU00090 IMP dehydrogenase dacA BSU00100 penicillin-binding protein 5*, D-alanyl-D-alanine carboxypeptidase pdxS BSU00110 pyridoxal-5'-phosphate synthase (synthase domain) pdxT BSU00120 pyridoxal-5'-phosphate synthase (glutaminase domain) serS BSU00130 seryl-tRNA-synthetase trnSL-Ser1 dck BSU00140 deoxyadenosin/deoxycytidine kinase dgk BSU00150 deoxyguanosine kinase yaaH BSU00160 general stress protein, survival of ethanol stress, SafA-dependent spore coat yaaI BSU00170 general stress protein, similar to isochorismatase yaaJ BSU00180 tRNA specific adenosine
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Energy Metabolism of Thehuman Fallopian Tube
    Energy metabolism of the human Fallopian tube I. A. Brewis, R. M. L. Winston and H. J. Leese department of Biology, University of York, Heslington, York Y01 5DD, UK; and 2 Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, DuCane Road, London W12 ONN, UK Summary. The consumption of oxygen (QO2), the production of lactate and the profile of four key metabolic enzymes were measured in small samples of human oviductal mucosa (endosalpinx) removed at surgery. The QO2 in the absence of substrate was 3\m=.\4\g=m\1O2 (mg dry wt) \m=-\1h\m=-\1,a value typical of quiescent tissue. The QO2 was stimulated by glucose, but diminished by glutamine and acetoacetate. Tissue lactate production was low and not increased by glucose. Hexokinase had the highest activity of the enzymes measured, followed by 2-oxoglutarate dehydrogenase; 6-phosphofructokinase and glycogen phosphorylase had low activities. The data are consistent with the proposition that glucose is a major metabolic fuel for human endosalpinx. Keywords: Fallopian tube; oxygen uptake; metabolism; glucose; lactate; human Introduction The human Fallopian tube consists of an outer muscle layer, the myosalpinx, which surrounds an inner mucosal lining, or endosalpinx. This paper reports data on the energy metabolism of the endosalpinx, the two specialized energy-requiring functions of which are the secretion of molecules into oviductal fluid (Leese, 1988) and the mechanical action of cilia. Endosalpinx from a variety of species including humans is being widely used in the 'co- culture' of preimplantation embryos (Bongso et ai, 1990). Information on the metabolism of the endosalpinx in vitro is likely to be important in defining its role in sustaining embryo development.
    [Show full text]
  • Zero-Shot Learning for Predicting Kinase Phosphorylation Sites
    DEEPKINZERO: ZERO-SHOT LEARNING FOR PREDICTING KINASE PHOSPHORYLATION SITES a thesis submitted to the graduate school of engineering and science of bilkent university in partial fulfillment of the requirements for the degree of master of science in computer engineering By Iman Deznabi August 2018 DeepKinZero: Zero-Shot Learning for Predicting Kinase Phosphoryla- tion Sites By Iman Deznabi August 2018 We certify that we have read this thesis and that in our opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Master of Science. Abdullah Ercüment Çiçek(Advisor) Öznur Taştan Okan (Co-advisor) Erman Ayday Ramazan Gökberk Cinbiş Approved for the Graduate School of Engineering and Science: Ezhan Karaşan Director of the Graduate School ii ABSTRACT DEEPKINZERO: ZERO-SHOT LEARNING FOR PREDICTING KINASE PHOSPHORYLATION SITES Iman Deznabi M.S. in Computer Engineering Advisor: Asst. Prof. Dr. A. Ercüment Çiçek and Asst. Prof. Dr. Öznur Taştan Okan August 2018 Protein kinases are a large family of enzymes that catalyze the phosphorylation of other proteins. By acting as molecular switches for protein activity, the phos- phorylation events regulate intracellular signal transduction, thereby assuming a central role in a broad range of cellular activities. On the other hand, aberrant kinase function is implicated in many diseases. Understanding the normal and malfunctioning signaling in the cell entails the identification of phosphorylation sites and the characterization of their interactions with kinases. Recent advances in mass spectrometry enable rapid identification of phosphosites at the proteome level. Alternatively, there are many computational models that predict phospho- sites in a given input protein sequence.
    [Show full text]
  • Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme a Kinase by Site-Directed Mutagenesis
    Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis Guneet Walia1, Komatireddy Gajendar2, Avadhesha Surolia1,2* 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, 2 National Institute of Immunology, New Delhi, India Abstract Dephosphocoenzyme A kinase performs the transfer of the c-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5–10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding.
    [Show full text]
  • 1787 Genome-Wide Identification and Characterization of Eukaryotic Protein Kinases Kanhu Charan Das1, Parismita Kalita1, Timir
    [Frontiers in Bioscience, Landmark, 25, 1787-1827, June 1, 2020] Genome-wide identification and characterization of eukaryotic protein kinases Kanhu Charan Das1, Parismita Kalita1, Timir Tripathi1 1Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Methods 3.1. F. gigantica gene sequence retrieval 3.2. Generation of hidden markov models (HMMs) 3.3. Classification and domain analysis of FgePKs 4. Results 4.1. Identification of kinase-like proteins in F. gigantica genome 4.2. Classification and domain analysis of FgePKs 4.2.1. Ser/Thr kinase group 4.2.1.1. cAMP-dependent protein kinase (AGC) group 4.2.1.1.1. Protein kinase C (PKC) 4.2.1.1.2. Protein kinase A (PKA) 4.2.1.2. Calmodulin kinase (CaMK) group 4.2.1.3. Cyclin-dependent kinase and other close relatives (CMGC) group 4.2.1.3.1. Cyclin dependent kinases (CDK) 4.2.1.3.2. Mitogen activated protein kinases (MAPK) 4.2.1.3.3. Glycogen synthase 3 kinases (GSK3) 4.2.1.3.4. Dual-specificity Tyr regulated kinases (DYRK) 4.2.1.4. Sterile Ser/Thr kinase (STE) group 4.2.1.5. Casein kinase 1 (CK1) group 4.2.2. Tyr kinase group 4.3.3. Hybrid group 4.3.3.1. Tyr kinase-like (TKL) group 4.3.3.2. Others 5. Discussion 6. Acknowledgments 7. References 1. ABSTRACT The tropical liver fluke, Fasciola gigantica is kinases (ePKs) are regulators of cellular a food-borne parasite responsible for the phosphorylation.
    [Show full text]