Metal cations defibrillize the amyloid beta-protein fibrils.

Amyloid beta-protein (A beta) is the major constituent of amyloid fibrils composing beta-amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A beta by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A beta 1-40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A beta 1-40 fibrils by metal cations was: Ca2+ and Zn2+ (IC50 = 100 microM) > Mg3+ (IC50 = 300 microM) > Al3+ (IC50 = 1.1 mM). EM analysis in negative staining showed that A beta 1-40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca2+, Mg2+, and Zn2+ to preformed A beta 1-40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al3+ was less effective, and reduced the fibril network by about 80% of that in the absence of any metal cation. Studies with A beta 1-42 showed that this peptide forms more dense network of fibrils as compared to A beta 1-40. Both ThT fluorescence spectroscopy and EM showed that similar to A beta 1-40, A beta 1-42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca2+. These studies suggest that metal cations can defibrillize the fibrils of synthetic A beta.

Phospholipid binding, phosphorylation by protein kinase C, and filament assembly of the COOH terminal heavy chain fragments of nonmuscle myosin II isoforms MIIA and MIIB.

Previously, we showed that myosin II heavy chains bind to phosphatidylserine (PS) liposomes via their COOH terminal regions and that protein kinase C (PK C) phosphorylates the PS-bound heavy chains [Murakami et al. (1994) J. Biol. Chem. 269, 16082-16090]. In this report, we studied the phospholipid binding, the kinetics of phosphorylation by PK C, and the effect of PK C-mediated phosphorylation on assembly using 46-47 kDa fragments from the COOH termini of macrophage (MIIAF46) and brain type (MIIBF47) heavy chain isoforms. Binding of the fragments to PS or phosphatidylinositol liposomes increased turbidity, but MIIAF46 gave higher turbidity than MIIBF47. Both fragments were sedimented similarly by ultracentrifugation in PS concentration and mole percent of PS dependent manners. With mixed PS/phosphatidylcholine (PC) liposomes, at least 70 mol % PS was required for heavy chain binding. A similar level of PS was required for phosphorylation of fragments by PK C, indicating that binding of tail regions to PS is a prerequisite for phosphorylation by PK C. PK C phosphorylated MIIBF47 with Vmax values 4-5 times higher than those of MIIAF46, but the Km values for the two substrates were similar. The apparent Km values for PS liposomes (Klipid) were also similar for phosphorylation of both isoforms. Mixing PS with PC increased the Klipid and reduced the Vmax values but did not alter the Km values for the substrates. Assembly of MIIBF47, but not MIIAF46, was significantly inhibited by the phosphorylation, indicating that nonmuscle myosin assembly can be regulated, in an isoform specific manner, via phosphorylation of heavy chains by PK C.

Effect of chronic hypo and hypervitaminosis C on the brush border enzymes and the intestinal uptake of glucose and alanine.

Brush border sucrase and alkaline phosphatase activities are considerably enhanced in the intestine of ascorbic acid deficient guinea-pigs. Similar increase in the uptake of D-glucose and L-alanine also occurs in chronic vitamin C deficiency. However the permeability of D-glucose and L-alanine in the intestine of animals fed with large doses of vitamin C is severely depressed, with a reduction in the levels of sucrase and alkaline phosphatase activities.