The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On electron microscopy, the final plasma membrane fractions are judged to be freethe basis of of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5'-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.     

Mechanical donor plasmapheresis using the Haemonetics Ultralite plasma collection system. 1. Collection of fresh plasma

In a clinical study altogether 449 machine donor plasmapheresis for collection of platelet poor fresh plasma were carried out using the new developed Haemonetics-Ultralite-Plasma-Collection-System. The average time of donation for 600 g of plasma amounted to 36 min. In comparison to other automated plasmapheresis-systems an effective plasma flow is recorded for the Ultralite-machine. All plasmapheresis procedures were well tolerated by the donors. The collected plasma is equivalent to the requirements for fresh frozen plasma. Complications due to the device or donors are small with 2.9% of the performed procedures.     

Significance of plasma skimming and plasma volume expansion.

The organs associated with plasma volume expansion, i.e., the red bone marrow, the enlarged spleen, and the uteroplacental complex, are arteriovenous shunts with an interposed sinusoidal stroma able to skim off plasma-rich blood. In the spleen, plasma separation is an integral part of the hemoconcentration. In the red bone marrow, plasma skimming might provide a washout mechanism for the many newly formed erythrocytes and platelets from the sinusoids to the peripheral blood circulation. In the uteroplacental complex, skimming of plasma-rich blood is beneficial in increasing blood flow in the myometrium, kidneys, and skeletal musculature. The hypervolemic status with anemia will simulate a negative iron balance, which speeds up the absorption of iron. Thus a conceptual unit seems to exist in which rheological factors influence such functions as transport of newly formed blood cells into the circulation (in the red bone marrow), hemoconcentration (in the spleen), and iron balance during pregnancy (in the uteroplacental complex).     

The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On the basis of electron microscopy, the final plasma membrane fractions are judged to be free of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5′-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.     

Effect of cold plasma on the E. coli cell wall and plasma membrane

The effect of cold plasma on E. coli cells was studied. It was shown that the treatment of E. coli cells with cold plasma caused partial or total disruption of the plasma membrane integrity, which was accompanied by a release of intracellular substances into the extracellular environment. A quantitative assessment of the extent of the damage to the cell membrane showed that a loss of no more than 23.6% of intracellular substances (calculated by the proportion of the intracellular nucleotide release) is sufficient to lead to cell death. The use of media with different ionic strength levels to create osmotic shock showed that the treatment of E. coli cells with cold plasma significantly decreased the cell wall strength.     

Electrostatic interactions at the plasma membrane.

Hydrogen-ion titration has been used to detect the presence of charged groups on the human red-cell plasma membrane. The findings are discussed in terms of the effect of the local environment on electrostatic interactions between the charged groups.     

The interaction of human plasma glycoaminoglycans with plasma lipoproteins.

Modifications of existing methods have allowed for the isolation and purification of various species of plasma glycosaminoglycans on the basis of their sulfate content and molecular size. All of the preparations precipitated human plasma low density lipoproteins (LDL); maximal precipitation occurred with amounts of glycans corresponding to 50 mug of hexuronate and 12 mg of LDL. The interaction of glycans with pyrene-labeled lipoproteins was also studied, measuring variations of the fluorescence emitted by the monomer (M) and excimer (E) species of the bound pyrene. The ratio IE/IM is proportional to c/eta, where c is the microscopic concentration of the pyrene confined to the hydrocarbon region of the lipoprotein and eta is the microviscosity of that region. To 0.12 mg of pyrene-labeled LDL, very low density lipoproteins (VLDL) or high density lipoproteins (HDL) were added increasing amounts of the various glycan preparations. The sulfate-rich species decreased the IE/IM ratio of LDL and HDL but not that of VLDL. This finding suggests that the glycan caused a change in lipoprotein conformation associated with either an increased volume or increased microscopic viscosity of the hydrocarbon region. The modification of LDL conformation could be prevented by proteolytic treatment of the sulfate-rich species or by addition to the system of suitable amounts of sulfate-poor species or of chrondroitin-4-sulfate, but could not be prevented by increased ionic concentration. These results suggest that the two main species of plasma glycans are important in maintaining adequate rheological properties of plasma lipoproteins.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane.

Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide (Mr 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel system for the analysis of membrane proteins is emphasized.     

Sugar transport by renal plasma membrane vesicles. Characterization of the systems in the brush-border microvilli and basal-lateral plasma membranes.

Uptake studies of D- and L-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brush-border vesicles D-glucose but not L-glucose transport was Na+ -dependent, was inhibited by phlorizin, and showed a transitory vesicle/medium ratio greater than 1, in the presence of an initial Na+ gradient. Basal-lateral membranes take up D-glucose faster than L-glucose, but the D-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorizin as compared to the brush-border fraction.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.