KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

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(1) A direct transfer mechanism for metabolites from the producing enzyme to the utilizing one via multienzyme complexes has been proposed for the glycolytic pathway. In view of its fundamental importance, we reinvestigated the evidence for direct transfer of NADH between its complexes with alpha-glycerol-3-phosphate dehydrogenase (GPDH) and with lactate dehydrogenase (LDH). Our results revealed that the proposed direct transfer of NADH between GPDH and LDH is mainly based on a misinterpretation of the experimental data, although some minor corrections of their data are also necessary. (2) A theory for explaining the effect of oscillations and energy-driven fluctuations on the environmental parameters on the catalytic activities of enzymes, particularly those embedded within cell membranes, has been formulated. The ability of proteins to absorb energy from oscillating external parameters into their conformational degree of freedom was emphasized. The theory provides a reasonable mechanism for energy transduction, even when the transduction is derived from a dynamic perturbation with a time average of zero. In addition, an oscillating electric pulse apparatus is being constructed for the purpose of experimentally verifying the above theory. (3) Affinity-purified anti-ubiquitin and anti-ubiquitin-BSA conjugates from sheep have been prepared and characterized. These antibodies were used to probe endogenous ubiquitin and ubiquitin-protein conjugates in various tissues, and used for immunocytochemical analysis of thin sections of brain. (4) Purification and characterization of ubiquitin activating enzyme from rabbit reticulocytes and human erythrocytes, and of isopeptidase are in progress. (5) Kinetics and mechanism of Fe(II)(ferrozine)3 formation from Fe(II)(amino acid)3 was investigated. A step wise mechanism was proposed and rate constants assigned. (6) Model study to examine the theory and practice for determining binding stoichiometry using the Job plot variation method was carried out using the Fe(II)-ferrozine system. The results revealed that for cooperative binding systems, anomalous stoichiometries can be obtained.