Mwt Stds, kD Following purification of the PYC enzyme by avidin-Sepharose affini

Question

Mwt Stds, kD Following purification of the PYC enzyme by avidin-Sepharose affinity chromatography, the investigators carried out several experiments to characterize the enzyme. First they ran samples of the enzyme on denaturing and non-denaturing gels. The results are shown in Figure 21.1. In addition, they ran the protein through a calibrated gel filtra- tion column. The results from the gel filtration column indicated that the PYC enzyme had a mo- lecular weight of 540 kilodaltons. Use this informa- Non-denaturing tion to determine the structure of the PYC enzyme 6. 272 132 95 45 Denaturing Figure 21.1: Gel electrophoresis of pyruvate carboxylase purified from M. thermo- autotrophicum. (Based on Mukhopadhyay, et al., 1998.)

Explanation / Answer

From Denaturing PAGE it is confirmed that the enzyme consists of two subunits of about A = 56 and B = 80 kDa, the non-denaturing gel confirms that size of the enzyme is around 135 kDa, which indicate the enzyme is in AB state (56 + 80 = 136 kDa) (each enzyme consists of 1 subunits of type A and 1 subunits of type B). The gel filtration data days PYC has a M.W. of 540 kDa, which indicate that enzyme form larger oligomer possibly of the type (AB)4, which corresponds to 135 x 4 = 540 kDa

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