Abstract
It is inevitable that most graduate students and researchers in the neurosciences will, from time to time, be faced with a problem related to enzymes. Tasks such as screening novel compounds for enzyme-inhibitor potency, examining the effects of drug administration on enzyme activities, employing enzymes as markers of disease state or cell fractionation, and elucidation of metabolic pathways for psychiatric drugs, are carried out on a regular basis. Yet few topics engender such anxiety and confusion for students of the neurosciences as does enzymology, with the consequence that relatively straightforward experiments can take weeks to complete, and results obtained are often misinterpreted or might even be meaningless. Most researchers doing enzyme assays are not enzymologists who deal with enzymes on a daily basis. Rather, they are following an assay protocol described in a manuscript or textbook, with no apparent necessity to understand the fundamentals of how enzymes and their inhibitors work in order to obtain results. More often than not, the researcher alters the original protocol slightly to suit the materials and apparatus available or to address a different question from that which the assay was designed to answer
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References
Brocklehurst, K (1996) Physical factors affecting enzyme activity, in Enzymology Labfax (Engel, P C, ed ) Academic, San Diego, CA, pp 175–198
Callmgliam, B A., Crosbie, A. E., and Rous, B A (1995) Some aspects of the pathophysiology of semicarbazide-sensitive amine oxidase enzymes Prog Brain Res. 106, 305–321.
Cheng, Y-C and Prusoff, W H (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22, 3099–3108
Clark, J. B (1993) Electrochemical assays: the oxygen electrode, in Enzyme Assays A practical approach (Eisenthal, R and Danson, M J., eds ) Oxford University Press, Oxford, UK, pp 181–190
Cleland, W W (1963a) The kinetics of enzyme-catalyzed reactions with two or more substrates or products I Nomenclature and rate equations Biochim Biophys Acta 67, 104–137
Cleland, W W (1963b) The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II Inhibition nomenclature and theory Biochim Biophys Acta 67, 173–187
Cleland, W W (1963c) The kinetics of enzyme-catalyzed reactions with two or more substrates or products. Ill Prediction of initial velocity and inhibition patterns by inspection Btochtm Btophys Acta 67, 188–196
Cornish-Bowden, A (1996) Classification and measurement of enzyme activity, in Enzymology Labfax (Engel, P C, ed) Academic, San Diego, CA, pp 1–7
Craig, D B, Arriaga, E A, Wong, J C. Y, Lu, H, and Dovichi, N J (1996) Studies on single alkaline phosphatase molecules reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation-the death of an enzyme J Am Chem Soc 118, 5245–5253
Dixon, M and Webb, E C. (1979) Enzymes Academic, New York
Dowd, J E and Riggs, D S (1965) A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations. J Biol Chem 240, 863–869
Eisenthal, R and Comish-Bowden, A (1974) The direct linear plot A new graphical procedure for estimating enzyme kinetic parameters Btochem J 139, 715–720
Engel, P C (1996a) Enzyme cofactors, in Enzymology Labfax (Engel, P C, ed ) Academic, San Diego, CA, pp 223–247
Engel, P C. (1996b) Enzyme kinetics, in Enzymology Labfax (Engel, P C, ed ) Academic, San Diego, CA, pp 77–113
Fowler, C J and Callingham, B. A (1979) The inhibition of rat heart type A monoamine oxidase by clorgyhne as a method for the estimation of enzyme active centers Mol Pharmacol 16, 546–555
Goldstein, A (1944) The mechanism of enzyme-inhibitor-substrate reactions illustrated by the cholmesterase-physostigmine-acetylcholine system J Gen Physiol 27, 529–580
Graham, J (1984) Isolation of subcellular organelles and membranes, in Cen-tnfugatton, 2nd ed A practical approach (Rickwood, D, ed ) Oxford University Press, Oxford, UK, pp 161–182.
Green, A L (1984) Assessment of the potency of reversible MAO inhibitors in vivo, in Monoamine Oxidase and Disease Prospects for Therapy with Reversible Inhibitors (Tipton, K F, Dostert, P and Strolin-Benedetti, M, eds ) Academic, London, pp 73–81
Henderson, P J F (1972) A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors Btochem J 127, 321–333
Henderson, P J F (1993) Statistical analysis of enzyme kmetic data, in Enzyme Assays A Practical Approach (Eisenthal, R. and Danson, M J, eds ) Oxford University Press, Oxford, UK, pp 277–316
Holt, A and Baker, G B (1995) Metabolism of agmatine (clonidine-displacing substance) by diamine oxidase and the possible implications for studies of imidazoline receptors Prog Brain Res 106, 187–197
Holt, A and Baker, G B (1996) Inhibition of rat brain monoamine oxidase enzymes by fluoxetine and norfluoxetine Naunyn-Schmiedeberg’s Arch Pharmacol 354, 17–24
Holt, A and Callingham, B A (1995) Further studies on the ex vivo effects of procarbazine and methylhydrazine on rat semicarbazide-sensitive amine oxidase and monoamine oxidase activities J Pharm Pharmacol 47, 837–845
Holt, A, Sharman, D F, Callingham, B A and Kettler, R (1992) Characteristics of procarbazine in vitro as an inhibitor of rat semicarbazide-sensitive amine oxidase J Pharm Pharmacol 44, 487–493
Holt, A, Sharman, D F, Baker, G B and Palcic, M M (1997) A continuous spectrophotometric assay for monoamine oxidase and related enzymes in tissue homogenates Ami Bwchem 244, 384–392
Iversen, L L (1963) The uptake of noradrenaline by the isolated perfused rat heart Br J Pharmacol 21, 523–537
King, E L and Altman, C (1956) A schematic method of deriving the rate laws for enzyme-catalyzed reactions J Phys Chem 60, 1375–1378
Kitz, R and Wilson, I. B. (1962) Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase J Biol. Chem 237, 3245–3249
Klinman, J P (1996) New quinocofactors in eukaryotes J Bwl Chem 271, 27189–27192.
Kornberg, A (1955) Lactic dehydrogenase of muscle, in Methods in Enzymology, vol 1, (Colowick, S. P and Kaplan, N O, eds) Academic, New York, pp 441–443
Lowe, C R and Thomas, J A (1996) Purification and analysis of enzyme preparations, in Enzymology Labfax (Engel, P. C, ed) Academic, San Diego, CA, pp 9–75
Lyles, G A. and Callingham, B A. (1982) In vitro and in vivo inhibition by benserazide of clorgyline-resistant amine oxidases in rat cardiovascular tissues Biochem Pharmacol 31, 1417–1424
Lyles, G A, Holt, A., and Marshall, C M S (1990) Further studies on the metabolism of methylamine by semicarbazide-sensitive amine oxidase activities in human plasma, umbilical artery and rat aorta J Pharm Pharmacol 42, 332–338
Morrison, J F (1982) The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions Trends Biochem Sci 1, 102–105
North, M J (1989) Prevention of unwanted proteolysis, in Proteolytic Enzymes A Practical Approach (Beynon, R J and Bond, J S, eds) Oxford University Press, Oxford, UK, pp. 105–124
Oldham, K G (1993) Radiometric assays, in Enzyme Assays A Practical Approach (Eisenthal, R and Danson, M J., eds ) Oxford University Press, Oxford, UK, pp. 93–122
Rang, H P, Dale, M M, and Ritter, J M (1995) Pharmacology Churchill Livingstone, New York
Segel, I H (1975) Enzyme Kinetics Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems Wiley, New York
Silverman, R B (1995) Mechanism-based enzyme inactivators, in Methods in Enzymology, vol 249 Enzyme Kinetics and Mechanism, Part D (Punch, D L, ed ) Academic, San Diego, CA, pp 240–283
Storer, A C, Darlison, M G, and Cornish-Bowden, A (1975) The nature of experimental error in enzyme kinetic measurements. Bwchem 151, 361–367
Straus, O H and Goldstein, A (1943) Zone behavior of enzymes illustrated by the effect of dissociation constant and dilution on the system cholinesterase-physostigmme J Gen Physiol 26, 559–585
Szedlacsek, S E. and Duggleby, R G. (1995) Kinetics of slow and tight-binding inhibitors, in Methods in Enzymology, vol 249 Enzyme Kinetics and Mechanism, Part D (Purich, D L, ed ) Academic, San Diego, CA, pp 144–180
Szutowicz, A, Kobes, R D, and Orsulak, P J (1984) Colonmetric assay for monoamine oxidase in tissues using peroxidase and 2,2’-azinodi (3-ethylbenzthiazoline-6-sulfonic acid) as chromogen Anal Biochem 138, 86–94
Tabor, C W, Tabor, H, and Rosenthal, S M (1954) Purification of amme oxidase from beef plasma J Bwl Chem 208, 645–661
Tipton, K F (1980) Kinetics and enzyme inhibition studies, in Enzyme Inhibitors as Drugs (Sandler, M, ed ) Macmillan, London, pp 1–23.
Tipton, K. F (1993) Principles of enzyme assay and kinetic studies, in Enzyme Assays A Practical Approach (Eisenthal, R and Danson, M J, eds ) Oxford University Press, Oxford, UK, pp 1–58.
Tipton, K F (1996) Patterns of enzyme inhibition, in Enzymology Labfax (Engel, P C, ed ) Academic, San Diego, CA, pp 115–174
Tunnicliff, G (1986) 4-Aminobutyrate transaminase, in Neuromethods, vol. 5, Neurotransmitter Enzymes (Boulton, A. A., Baker, G. B, and Yu, P H, eds ) Humana, Clifton, N J., pp 389–419.
Weitzman, P. D J and Watkins, P. J. (1993) Electrochemical assays polarogra-phy, in Enzyme Assays A Practical Approach (Eisenthal, R. and Danson, M J, eds ) Oxford University Press, Oxford, UK, pp 167–179
Williams, J W and Morrison, J. F (1979) The kinetics of reversible tight-binding inhibition, in Methods in Enzymology, vol 63 Enzyme Kinetics and Mechanism, Part A (Punch, D. L, ed ) Academic, San Diego, CA, pp. 437–467.
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Holt, A. (1999). On the Measurement of Enzymes and their Inhibitors. In: Cell Neurobiology Techniques. Neuromethods, vol 33. Humana Press. https://doi.org/10.1385/0-89603-510-7:131
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DOI: https://doi.org/10.1385/0-89603-510-7:131
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