Constitutive Inorganic Pyrophosphatase of Escherichia coli: III. MOLECULAR WEIGHT AND PHYSICAL PROPERTIES OF THE ENZYME AND ITS SUBUNITS

Abstract

Abstract The hydrodynamic properties of native Escherichia coli pyrophosphatase in 0.1 m NaCl, 0.01 m sodium phosphate buffer, pH 7, were as follows: s20,w0 = 7.01 S, D20,w0 = 5.70 x 10-7 cm2 sec-1 (molecular weight by sedimentation-diffusion = 118,000), and [η] = 4.00 cc per g. The molecular weights obtained with high and low speed sedimentation equilibrium techniques were 122,000 and 119,000, respectively. Optical rotatory dispersion measurements gave evidence of little or no α helix structure; there was a small trough at 230.5 mµ. In studies cited in the Appendix, electron micrographs of negatively stained preparations of the enzyme revealed a round object 65 A in diameter. If this object represents a spherical particle, its estimated weight is 117,000 daltons. In mother liquor from which the enzyme had been recrystallized there were, in addition to the 65 A forms, larger, round objects 130 to 140 A in diameter. As the solution pH was lowered from 7 to 5, increasing numbers of these aggregates appeared and at the expense of 65 A particles. When the protein was denatured in 5 m guanidine hydrochloride (GuHCl) and either alkylated with N-ethylmaleimide or kept reduced with dithiothreitol, the following properties were observed: s20,guhcl0 = 0.595 S, D20,guhcl0 = 3.9 x 10-7 cm2 sec-1 (molecular weight by sedimentation-diffusion = 21,300), and [η] = 21.8 cc per g. The molecular weights determined with high and low speed sedimentation equilibria were 20,600 and 18,300, respectively. The optical rotatory dispersion was similar to that of native enzyme except that the 230.5 mµ trough was abolished. It is concluded that the enzyme is a compact, globular protein consisting of 6 subunits of molecular weight 20,000 each. In 5 m guanidine hydrochloride the subunits behave as randomly coiled, single polypeptide chains.