SUMMARYIn reconstituted casein systems, complete sub-micelles were previously observed only under extreme conditions of ionic strength, namely a NaCl concentration of 2 M or greater. However, studies with the analytical ultracentrifuge on phosphate-containing casein systems revealed that under certain conditions, a stable sub-micelle-micelle equilibrium was established. Conditions which were standard throughout were 7.5 mg/ml protein, 0.04 M-NaCl, pH 6.6 and 37 °C. The concentrations of added CaCl2 (Ca2+) and inorganic phosphate (P1) were variable. With no P1 present, Ca-sensitive caseins precipitated or formed micelles when k-casein was present, between 6 and 7 mM-Ca2+. With 10–20 mM-P1 precipitation or micelle formation began at about 4 mM-Ca2+ and was complete at about 5 mM-Ca2+. Over this interval, during micelle formation, a sub-micelle peak with s20, w of about 13 S was observed in equilibrium with the broad micelle peak. In a system containing a 2:2:1 weight ratio of ±sI-:²-:k-casein, this sub-micelle was isolated at 4 mM-Ca2+ and 2.5 mM-P1. The mol. wt was 760000 and it thus contained approximately 33 monomer caseins. The reconstituted micelle system formed in the absence of P1 was quite temperature sensitive, forming at 37 °C but disappearing upon cooling. In the presence of P1 the micelles formed at 37 °C but were stable to cooling as are natural micelles. Evidently, a combination of hydrophobic and electrostatic interactions are involved in natural micelle formation, probably with the production of salt bridges of Ca and phosphate ions between sub-micelles.
Dual Mechanism of Ion Permeation through VDAC Revealed with Inorganic Phosphate Ions and Phosphate Metabolites