Phosphofructokinase on Contraction and Relaxation in Rabbit Skeletal

Muscle (Part 2, Effect of ATP on Phosphofructokinase)

Abstract

In our previous paper1), we reported that phosphofructokinase (PFK) in a Trissoluble form is converted into a Tris-insoluble form upon relaxation or contraction of the muscle, and that this conversion might be presumably mediated by ATP. The present studies were undertaken in an attempt to elucidate what a Tris-soluble form and an insoluble one are, and also what role ATP plays in the conversion mechanism.
The partially purified enzyme1), one ml of which contained the enzyme prepared from 4 w. w. g. of the rabbit backmuscle (white muscle), was dialysed at 4° for 90 minutes against Tris-HCI (20 mM, pH 8.0) buffer with mercaptoethanol (MSH-10 mM). The activities of the enzyme dialysed decreased at 30°. However, addition (5 mM) of MgSO₄ or (NH₄)₂SO₄ instead of NH₄Cl protected the decrease in activities. This indicates that SO₄^-- is a stabilizing factor for this enzyme.
By using Tris-maleate buffer (pH 5.5-8.5) instead of Tris-HCl, the dialysed enzymes were centrifuged at 10⁴ rpm for 10 minutes. The activities after activation (preincubation with ATP at 30°C for 30 minutes, pH 8.0) in the supernatants (sup) and precipitants (ppt) were measured. It was demonstrated that the activities in the sup decreased, while those in the ppt increased according to lowering of pH, and that the enzymes dialysed at pH 5.5 completely precipitated. The enzymes were extracted from ppt by a buffer containing ATP (2 mM) or MgSO₄(10 mM) at pH 5.5, and also by a buffer containing some stabilizing factors at pH 8.0.
The activities dependent on pH were examined in Tris-maleate buffer including MgSO₄ (10 mM) or ATP (2 mM), in which the enzymes were soluble even at a low pH. In the buffer including ATP, the activities at pH 5.5 were maintained at the same value as at pH 8.0. In the buffer including MgSO₄, the activities were lost at pH 5.5, although the value at pH 8.0 was equivalent to one when ATP was added. But the activities after activation showed an equal highest value in both buffers at pH 5.5-8.5.
In Sephadex G-200 column chromatography using the buffer at pH 8.0 including MgSO₄ as an eluant, the enzyme appeared in a high molecular fraction with the activities as PFK. By using the buffer at pH 5.5, the enzyme appeared in a low molecular fraction, of which the activities as PFK were lost but appeared after activation. By using the buffer at pH 5.5 including ATP instead of MgSO₄, the enzyme appeared in a high molecular fraction with the activities as PFK. Under the same conditions as described above, S_20W of the purified enzymes (from Boehringer Mannheim GmbH-Mannheim, Germany) were 14.3S, 5.4S and 22.5S, respectively.
The ppt at pH 5.5 was suspended in K₂HPO₄ (50 mM) and MSH(10 mM), or in Tris-maleate (20 mM, pH 8.0) and MSH (10 mM) including MgSO₄ (10 mM) or ATP (2 mM), and then these activities immediately followed. The result was that every suspension showed a gradual increase in activities. On the other side, when the ppt was suspended only by Tris-maleate buffer, the activities appeared only by additions of ATP or ADP, but not by AMP, K₂HPO₄ or MgSO₄.
From these studies, it was concluded that phosphofructokinase was insoluble by decreasing amounts of ATP and lowering of pH(muscle contraction) and soluble by increasing amounts of ATP and ascending of pH (muscle relaxation), and that the activities of this enzyme were regulated by conversion between the inactive(5S) form and the active (14S-22S) one, and also that this enzyme was solubilized and aggregated to the active form by ATP even at a low pH and, in addition, might be mediated specifically by ATP-probably phosphorylation of protein.