NADH-glutamate dehydrogenase (GDH) is active in human tissues, and is chromatographically purified, and studied because it participates in synthesizing glutamate, a neurotransmitter. But chromatography dissociates the GDH isoenzymes that synthesize nongenetic code-based RNA enzymes degrading superfluous mRNAs thereby aligning the cellular reactions with the environment of the organism. The aim was to electrophoretically purify human hexameric GDH isoenzymes and to characterize their RNA enzyme synthetic activity as in plants. The outcome could be innovative in chemical dependency diagnosis and management. Multi metrix electrophoresis including free solution isoelectric focusing, and through polyacrylamide and agarose gels were deployed to purify the redox cycle isoenzymes of laryngeal GDH, and to assay their RNA enzyme synthetic activities. The laryngeal GDH displayed the 28 binomial isoenzymes typical of higher organisms. Isoelectric focusing purification produced pure GDH. Redox cycle assays of the GDH isoenzymes produced RNA enzymes that degraded human stomach total RNA. In the reaction mechanism, the Schiff-base intermediate complex between α-ketoglutarate and GDH is the target of nucleophiles, resulting to the disruption of synthesis of glutamate, and RNA enzyme. The strongest nucleophiles are the psychoactive alkaloids of tobacco, cocaine, opium poppy, cannabis smoke because they are capable of reacting with GDH Schiff base intermediate to stimulate synthesis of aberrant RNA enzymes that degrade cohorts of mRNAs thereby changing the biochemical pathways and exacerbating drug overdose and chemical dependency. Electrophoretic purification, and characterization of the RNA enzyme synthetic activity set the forecourt for innovative application of GDH redox cycles in the diagnostic management of chemical dependency.
References
[1]
Burbaeva, G.S., Boksha, I.S., Turishcheva, M.S., Vorobyeva, E.A., Savushkina, O.K. and Tereshkina, E.B. (2003) Glutamine Synthetase and Glutamate Dehydrogenase in the Prefrontal Cortex of Patients with Schizophrenia. Progress in Neuro-Psychopharmacology Biological Psychiatry, 27, 675-680. https://doi.org/10.1016/S0278-5846(03)00078-2
[2]
Malthankar-Phatak, G.H., De Lanerolle, N., Eid, T., Spencer, D.D., Behar, K.L., Spencer, S.S., Kim, J.H. and Lai, J.C.K. (2006) Differential Glutamate Dehydrogenase (GDH) Activity Profile in Patients with Temporal Lobe Epilepsy. Epilepsia, 47, 1292-1299. https://doi.org/10.1111/j.1528-1167.2006.00543.x
[3]
Kanamori, K. and Ross, B.D. (1995) Steady-State in Vivo Glutamate Dehydrogenase Activity in Rat Brain Measured by 15N NMR. Journal of Biological Chemistry, 270, 24805-24809. https://doi.org/10.1074/jbc.270.42.24805
[4]
Tanizawa, Y., Nakai, K., Sasaki, N., Anno, T., Ohta, Y., Inoue, H., Matsuo, K., Koga, M., Furukawa, S. and Oka, Y. (2002) Unregulated Elevation of Glutamate Dehydrogenase Activity Induces Glutamine-Stimulated Insulin Secretion. Diabetes, 51, 712-717. https://doi.org/10.2337/diabetes.51.3.712
[5]
Shashidharan, P., Michaelidis, M., Robakis, N.K., Kresovalis, A., Papamatheakis, J. and Plaitakis, A. (1994) Novel Human GDH Expressed in Neural and Testicular Tissues and Encoded by X-Linked Intronless Gene. Journal of Biological Chemistry, 269, 16971-16976. https://doi.org/10.1016/S0021-9258(19)89484-X
[6]
Burbaeva, G.S., Turishcheva, M.S., Vorobyeva, E.A., Savushkina, O.K., Tereshkina, E.B. and Boksha, I.S. (2002) Diversity of Glutamate Dehydrogenase in Human Brain. Progress in Neuro-Psychopharmacology Biological Psychiatry, 26, 427-435. https://doi.org/10.1016/S0278-5846(01)00273-1
[7]
Lee, W.-K., Shin, S., Cho, S.S. and Park, J.-S. (1999) Purification and Characterization of Glutamate Dehydrogenase as Another Isoprotein Binding to the Membrane of Rough Endoplasmic Reticulum. Journal of Cellular Biochemistry, 76, 244-253. https://doi.org/10.1002/(SICI)1097-4644(20000201)76:2<244::AID-JCB8>3.0.CO;2-K
[8]
O’Carra, P., Griffin, T., O’Flaherty, M., Kelley, N. and Mulcahy, P. (1996) Further Studies on the Bioaffinity Chromatography of NAD+-Dependent Dehydrogenases Using Locking-on Effect. Biochimica et Biophysica Acta, 1297, 235-243. https://doi.org/10.1016/S0167-4838(96)00100-8
[9]
Smith, T.J. and Stanley, C.A. (2012) Chapter 1. Glutamate Dehydrogenase. In: D’Mello, J.P.F., Ed., Amino Acids in Human Nutrition and Health, CAB International Publisher, Oxfordshire, 3-23. https://doi.org/10.1079/9781845937980.0003
[10]
Cammaerts, D. and Jacobs, M. (1983) A Study of the Polymorphism and the Genetic Control of the Glutamate Dehydrogenase in Arabidopsis Thaliana. Plant Science Letters, 31, 65-73. https://doi.org/10.1016/0304-4211(83)90130-X
[11]
Osuji, G.O. and Braithwaite, C. (1999) Signaling of Glutamate Dehydrogenase in Response to Pesticide Treatment and Nitrogen Fertilization of Peanut (Arachis hypogaea L). Journal of Agricultural and Food Chemistry, 47, 3332-3344. https://doi.org/10.1021/jf9805303
[12]
Osuji, G.O. and Madu, W.C. (1995) Ammonium Ion Dependent Isomerization of Glutamate Dehydrogenase in Relation to Glutamate Synthesis in Maize. Phytochemistry, 39, 495-503. https://doi.org/10.1016/0031-9422(94)00976-Z
[13]
Osuji, G.O. and Madu, W.C. (2015) Chapter 1. Glutamate Dehydrogenase. In: D’Mello, J.P.F., Ed., Amino Acids in Higher Plants, CAB International Publisher, Oxfordshire, 1-29. https://doi.org/10.1079/9781780642635.0001
[14]
Osuji, G.O., Braithwaite, C., Pointer, R. and Reyes, J. (1999) Pesticide Inactivation of Peanut Glutamate Dehydrogenase: Biochemical Basis of the Enzyme’s Isomerization. Journal of Agricultural and Food Chemistry, 47, 3345-3351. https://doi.org/10.1021/jf980531v
[15]
Fersht, A. (1985) Chemical Catalysis. In: Enzyme Structure and Mechanisms, 2nd Edition, W H Freeman & Co, New York, 47-97.
[16]
Osuji, G.O., Johnson, P.M. and Madu, W.C. (2021) Optimization of Cowpea dry Grain Yield through the Stimulation of the RNA Synthetic Activity of NADH-Glutamate Dehydrogenase. American Journal of Plant Sciences, 12, 71-103. https://doi.org/10.4236/ajps.2021.121006
[17]
Osuji, G.O., Madu, W.C. and Johnson, P.M. (2019) Total RNA Degradation in Vitro and in Vivo by Glutamate Dehydrogenase-Synthesized RNA Enzyme: Biotechnological Applications. Advances in Bioscience and Biotechnology, 10, 59-85. https://doi.org/10.4236/abb.2019.104005
[18]
Osuji, G.O. and Johnson, P.M. (2018) Structural Properties of the RNA Synthesized by Glutamate Dehydrogenase for the Degradation of Total RNA. Advances Enzyme Research, 6, 29-52. https://doi.org/10.4236/aer.2018.63004
[19]
U.S. DHHS (2010) How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease. United States Department of Health Human Services: A Report of the Surgeon General, Rockville.
[20]
UNODC (2012) Recommended Methods for the Identification and Analysis of Cocaine in Seized Materials. Laboratory and Scientific Section, United Nations Office on Drug and Crime, Vienna. United Nations, New York.
[21]
Schwilke, E.W., Schwope, D.M., Karschner, E.L., Lowe, R.H., Darwin, W.D., Kelley, D.L., et al. (2009) Δ9-Tetrahydrocannabinol (THC), 11-Hydroxy-THC, and 11-Nor-9-Carboxy-THC Plasma Pharmacokinetics during and after Continuous High-Dose oral THC. Clinical Chemistry, 55, 2180-2189. https://doi.org/10.1373/clinchem.2008.122119
[22]
Mozaffarian, D. (2020) Dietary and Policy Priorities to Reduce the Global Crises of Obesity and Diabetes. Nature Food, 1, 38-50. https://doi.org/10.1038/s43016-019-0013-1
[23]
Zheng, F. and Zhan, C. (2012) Modeling of Cocaine in Human Reveals the Feasibility for Development of Enzyme Therapies for Drugs of Abuse. PLoS Computational Biology, 8, e1002610. https://doi.org/10.1371/journal.pcbi.1002610
[24]
Flygare, S., Griffin, T., Larsson, P.-O. and Mosbach, K. (1983) Affinity Precipitation of Dehydrogenases. Analytical Biochemistry, 133, 409-416. https://doi.org/10.1016/0003-2697(83)90102-1
[25]
McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Purification of Glutamate Dehydrogenase from Ox Brain and Liver. Evidence That Commercially Available Preparations of the Enzyme from Ox Liver Have Suffered Proteolytic Cleavage. Biochemical Journal, 191, 605-611. https://doi.org/10.1042/bj1910605
[26]
Godinot, C., Julliard, J.H. and Gautheron, D.C. (1974) A Rapid and Efficient New Method of Purification of Glutamate Dehydrogenase by Affinity Chromatography on GTP-Sepharose. Analytical Biochemistry, 61, 264-270. https://doi.org/10.1016/0003-2697(74)90353-4
[27]
Preiss, T., Hall, A.G. and Lightowlers, R.N. (1993) Identification of Bovine Glutamate Dehydrogenase as an RNA Binding Protein. Journal of Biological Chemistry, 268, 24523-24526. https://doi.org/10.1016/S0021-9258(19)74494-9
[28]
Brodelius, P.E. and Kaplan, N.O. (1979) Studies of Bovine Liver Glutamate Dehydrogenase by Analytical Affinity Chromatography on Immobilized AMP Analogs. Archives of Biochemistry and Biophysics, 194, 449-456. https://doi.org/10.1016/0003-9861(79)90639-8
[29]
Osuji, G.O., Konan, J. and M’Mbijjewe, G. (2004) RNA Synthetic Activity of Glutamate Dehydrogenase. Applied Biochemistry and Biotechnology, 119, 209-228. https://doi.org/10.1007/s12010-004-0003-z
[30]
Laemmli, U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227, 680-685. https://doi.org/10.1038/227680a0
[31]
Merril, C.R., Goldman, D. and Van Keuren, M.L. (1984) Gel Protein Stain. Methods in Enzymology, 104, 441-447. https://doi.org/10.1016/S0076-6879(84)04111-2
[32]
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein Measurement with the Folin-Phenol Reagent. Journal of Biological Chemistry, 193, 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6
[33]
Osuji, G.O., Mangaroo, A.S. and Roberts, P.S. (2001) In Vitro Isomerization of Glutamate Dehydrogenase in Relation to Phytosequestration of Lead. Southern Association Agricultural Scientists Bulletin: Biochemistry and Biotechnology, 14, 60-72.
[34]
Osuji, G.O. and Madu, W.C. (1997) Regulation of Peanut Glutamate Dehydrogenase by Methionine Sulphoximine. Phytochemistry, 46, 817-825. https://doi.org/10.1016/S0031-9422(97)00395-6
[35]
Hammond, S., Caudy, A.A. and Hammond, G.J. (2001) Post-Transcriptional Gene Silencing by Double-Stranded RNA. Nature Reviews/Genetics, 2, 110-119. https://doi.org/10.1038/35052556
[36]
Zamore, P.D., Tuschl, T., Sharp, P.A. and Bartel, D.P. (2000) RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21-23 Nucleotide Intervals. Cell, 101, 25-33. https://doi.org/10.1016/S0092-8674(00)80620-0
[37]
Cogoni, C. and Macino, G. (2000) Post-Transcriptional Gene Silencing across Kingdoms. Current Opinion Genetics & Development, 10, 638-643. https://doi.org/10.1016/S0959-437X(00)00134-9
[38]
McKenzie, A. and Kolb, A. (2021) The Human Genome at 20: How Biology’s Most-Hyped Breakthrough Led to Anticlimax and Arrests. https://phys.org/news/2021-02-human-genome-biology-most-hyped-breakthrough.html
[39]
Osuji, G.O., Duffus, E., Johnson, P., Woldesenbet, S., Weerasooriya, A., Ampim, P.A., Carson, L., Jung, Y., South, S., Idan, E., Johnson, D., Clarke, D., Lawton, B., Parks, A., Fares, A. and Johnson, A. (2015) Enhancement of the Essential Amino Acid Composition of Food Crop Proteins through Biotechnology. American Journal of Plant Sciences, 6, 3091-3108. https://doi.org/10.4236/ajps.2015.619302
[40]
Osuji, G.O., Brown, T.K., South, S.M., Johnson, D. and Hyllam, S. (2012) Molecular Modeling of Metabolism for Allergen-Free Low Linoleic Acid Peanuts. Applied Biochemistry and Biotechnology, 168, 805-823. https://doi.org/10.1007/s12010-012-9821-6
[41]
Osuji, G.O., Brown, T.K., South, S.M., Duncan, J.C. and Johnson, D. (2011) Doubling of Crop Yield through Permutation of Metabolic Pathways. Advances in Bioscience and Biotechnology, 2, 364-379. https://doi.org/10.4236/abb.2011.25054
[42]
Hwang, D., Rust, A.G., Ramsey, S., Smith, J.J., Leslie, D.M., Weston, A.D., et al. (2005) A Data Integration Methodology for Systems Biology. Proceedings of the National Academy of Sciences of the United States of America, 102, 17296-17301. https://doi.org/10.1073/pnas.0508647102
[43]
Burke, P.E.P., de L. Campos, C.B., de F. Costa, L. and Quiles, M.G. (2020) A Biochemical Network Modeling of a Whole-Cell. Scientific Reports, 10, Article No. 13303. https://doi.org/10.1038/s41598-020-70145-4
