Date of Award

1985

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Rights

CC BY-NC-ND 4.0

Abstract

Both glucose- and succinate-grown whole cells of P. putida transport 2-keto-D-gluconate (2KGA) by a saturable process. The transport of 2KGA by membrane vesicles prepared from glucose-grown P. putida occurs by a facilitated diffusion process, with an apparent Km of 111.0 (+OR-) 2.9 (mu)M and a Vmax of 0.55 (+OR-) 0.04 nmol.mg protein.('-1)min.('-1). The provision of artificial and natural electron donors results in a significant decrease in Km value, while the Vmax remains virtually unchanged. 2KGA transport is inhibited by antimycin A, rotenone and DNP in the presence of L-malate/FAD. 4F2KGA and 3F2KGA also inhibit 2KGA transport competitively. With vesicles prepared from succinate-grown cells, 2KGA transport occurs by a non-specific diffusion process. Vesicles prepared from glucose- or succinate-grown cells generate a proton-motive force ((DELTA)p) of -145 mV and -140 mV, respectively, when provided with PMS/ascorbate. Both the (DELTA)pH and (DELTA)(psi) components are collapsed in the presence of DNP. In the presence of L-malate/FAD, a (DELTA)p of -129 mV is generated by vesicles from glucose-grown cells, while no (DELTA)p is detected with vesicles from succinate-grown cells. The active transport of 2KGA by vesicles from glucose-grown cells is coupled predominantly to the (DELTA)pH component of (DELTA)p at pH 6.6. L-Malate is transported by a non-specific diffusion process in vesicles from succinate-grown cells however, L-malate transport and L-malate induced proton uptake display saturation kinetics with Km values of 14.3 mM and 16.0 mM, respectively. The Vmax values are 313 nmol.mg protein.('-1) min.('-1) for L-malate uptake and 667 nmol.mg protein.('-1) min.('-1) for L-malate induced proton uptake. During L-malate transport, oxaloacetate is produced extra-cellularly, while succinate appears to accumulate intra-cellularly. Based on these observations the transport of L-malate in this organism is considered to be more elaborate than hitherto documented.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1985 .A433. Source: Dissertation Abstracts International, Volume: 46-02, Section: B, page: 0505. Thesis (Ph.D.)--University of Windsor (Canada), 1985.

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