Double Membrane Filtration Research Articles

Selective removal of low density lipoprotein by plasmapheresis in familial hypercholesterolemia.

Plasma lipoproteins were selectively removed from familial hypercholesterolemic patients by using two types of plasmapheresis: double-membrane filtration and selective adsorption of very low density lipoproteins (VLDL) and low density lipoproteins (LDL). In both techniques, plasma was separated from blood cells by using hollow-fiber filters, and 100% of the VLDL and LDL was recovered in the filtrate. In double-membrane filtration, the second hollow-fiber filter trapped 84% of LDL + VLDL, 48% of high density lipoprotein (HDL), 24% of albumin, and 46% of the remaining plasma protein. By treating 3 liters of plasma from a patient weighing 60 kg, 60% of the LDL and 30% to 40% of the HDL were removed as a result of an exponential decay of each component with the respective trapping coefficients. When dextran sulfate-cellulose was used as a LDL sorbent, there was only loss of LDL and VLDL, and no loss of any other major plasma component or of HDL. The sorbent column (400 ml) was saturated with 7.5 g of LDL cholesterol by treatment with 3.5 liters of plasma; the maximum reduction of LDL cholesterol was thus about 300 mg/dl for the patient weighing 60 kg. No serious side effects were observed during the long-term trials (19 to 27 months for four patients on double-membrane filtration and 10 months for the two patients on dextran sulfate-cellulose treatment.

Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+,Mg2+-dependent ATPase of sarcoplasmic reticulum.

The amount of Ca2+ bound to the Ca2+,Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509--523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase. In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2. Under the conditions where most EP and ADP sensitive at steady state (58 microM Ca2+, 50 microM EGTA, and 20 mM MgCl2 at pH 7.0 and 0 degrees C), bound Ca2+ increased by 0.6--0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2+ + EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one. Under the conditions where most EP was ADP insensitive at steady state (58 microM Ca2+, 30 microM EGTA, and 20 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 microM Ca2+, 25 microM EGTA, and 1 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ did not change upon addition of ATP. These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.