Search PubMed⌕ Search

Biomedical subjects

T J Mueller

Publications and source records attributed to T J Mueller.

At least 37 records · Page 2Linked to original sources
PubMed1984

Hydrogenation of triton X-100 eliminates its fluorescence and ultraviolet light absorption while preserving its detergent properties.

The ultraviolet-light absorption and fluorescence of Triton X-100 were virtually eliminated by hydrogenation to its reduced cyclohexyl analog, RTX-100. The critical micelle concentration of RTX-100 was 12% higher than that of Triton X-100. RTX-100 and Triton X-100 were quite similar in their abilities to extract proteins from human erythrocyte membranes.

Chromatography, Thin Layer↗
PubMed1983

Does cell density correlate with red cell age?

We have examined age-related changes in red cells by employing a serial hypertransfusion protocol to generate populations of in vivo-aged mouse red cells. Studies of these old cells have revealed alterations in membrane components; namely, a conversion of band 4.1b to 4.1a and an increase in the amount of membrane-associated, Triton-insoluble globin. Furthermore, we have found that the erythrocyte does change in density, but only during the earliest stages of its life-span. As the cell continues to age, there is no longer a correlation between age and density.

Animals↗
PubMed1981

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

Azotobacter↗
PubMed1981

On the hemolytic and thrombogenic potential of occluder prosthetic heart valves from in-vitro measurements.

An experimental investigation was conducted to determine the magnitude of shear stresses and areas of stasis of several types of prosthetic occluder heart valves. These experiments were performed in a steady-flow test loop using an axisymmetric aortic-shaped test chamber and an aqueous-glycerine solution. The flow loop produced a low-turbulence intensity and uniform mean velocity profile upstream of the test chamber. Tests were performed on a Kay-Shiley disk, a Bjork-Shiley tilting disk and Starr-Edwards Models 1260 and 2320 ball prostheses at Reynolds numbers between 2000 and 6200. Momentum transfer and turbulence data were obtained both around and distal to the valve occluders using laser Doppler and hot-film anemometry. The region directly surrounding the valve occluders contained the largest stresses measured. Aortic wall shear measurements revealed magnitudes potentially damaging to the vessel lining. Regions of slowly moving separated flow found to exist in these occluder valve flow fields correlated with clinical findings of thrombus formation.

Aorta↗
PubMed1981

Protein architecture of the erythrocyte membrane.

The normal human erythrocyte is comprised of as many as 90 polypeptides. These membrane polypeptides are organized asymmetrically within the membrane. In a well-washed erythrocyte, all the polypeptides exposed on the outside surface are transmembrane proteins. Six such polypeptides have been identified. They are the anion transport component protein 3, the glucose transport protein 4.5 and the sialoglycoproteins PAS 1, 2', 2 and 3. The major membrane protein, protein 3, which comprises 25% of the total membrane peptide, interacts on the cytoplasmic surface with the cytoskeletal components. The sialoglycoprotein PAS 2 has also been shown to interact with cytoskeleton and has been named glycoconnectin. Employing a monolayer freeze-fracture technique, the transmembrane proteins have been shown to distribute asymmetrically. For example, the major sialoglycoproteins distribute with the outer half of the bilayer (E-face) while protein 3 is found exclusively on the inner half of the bilayer (P-face). The anchoring of the transmembrane proteins determines on which half of the bilayer the protein will be found. Well-defined fragments of the sialoglycoprotein are produced by the freeze-fracture procedure indicating that selected covalent bonds of these transmembrane proteins were broken. Correlation of these results with the appearance of intramembrane particles on the E- and P-faces indicate that protein 3 may account for most of the intramembrane particles found on the P-face, while the sialoglycoproteins probably account for little, if any, of the particles. Although the evidence is not conclusive, protein 4.5 may account for the particles on the E-face of the freeze-fractured human erythrocyte membrane.

Blood Proteins↗
PubMed1981

Glycoconnectin (PAS 2), a membrane attachment site for the human erythrocyte cytoskeleton.

The sialoglycoprotein PAS 2 is present in cytoskeletons generated by Triton X-100 extraction of isolated human erythrocyte stroma. However, removal of the peripheral cytoskeletal proteins by elution with 0.1N NaOH prior to Triton extraction renders PAS 2 Triton-soluble. This suggests association of PAS 2 with the cytoskeletal elements lining the inner surface of the erythrocyte membrane. For this reason, we are proposing the name glycoconnectin for PAS 2, since it is a glycoprotein which connects the core of the cytoskeleton to the membrane bilayer. The cytoskeletal proteins bands 4.1a,b also appear to interact directly with the membrane, since all of the other peripheral membrane proteins can be eluted with NaOH, pH 11.5, without releasing bands 4.1a,b from the membrane. Removal of spectrin and actin from the membrane results in the solubilization of both glycoconnectin and bands 4.1a,b by Triton X-100. Glycoconnectin is not present in the cytoskeletons derived from a donor whose membranes are devoid of bands 4.1a,b. These data suggest that glycoconnectin may interact directly with bands 4.1a,b.

Blood Proteins↗
PubMed1979

Effect of endo-beta-galactosidase on intact human erythrocytes.

Endo-beta-galactosidase, a glycosidase that hydrolyzes Gal beta 1-4 GlcNAc linkages in glycoconjugates, has been used to probe the plasma membrane of human erythrocytes. Coomassie blue staining of stroma components separated by sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that treatment of red cells with endo-beta-galactosidase converts Protein 3, the anion transporter of the erythrocyte, to a more compact staining band. No other components detected by Coomassie staining are affected. Following labeling of red cells with galactose oxidase + NaB3H4, 45 to 50% of the [3H]galactose residues can be released by endo-beta-galactosidase. In contrast, only 5% of the label incorporated by treatment with periodate + NaB3H4, can be removed. [3H]Galactose residues are released from three components: Protein 3, Band 4.5, and the megaloglycolipids. The susceptibility of these components to endo-beta-galactosidase, together with the high content of Gal and GlcNAc present in Protein 3 and the megaloglycolipids, suggests that the erythrocyte membrane contains several components with N-acetyllactosamine repeating units, a structure commonly found in connective tissue glycoconjugates.

Erythrocyte Membrane↗
PubMed1979

Distribution of transmembrane polypeptides in freeze fracture.

Human erythrocytes have been freeze-fractured, and the polypeptides associated with the separate halves of the membrane bilayer have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The transmembrane proteins were differentially separated by the fracture process. Although sialoglycoproteins associated with the outer half of the membrane, the anion transport protein (band 3) mainly remained with the inner half of the membrane. Well-defined fragments of the sialoglycoproteins were produced by the freeze-fracture procedure, indicating that selected covalent bonds of these transmembrane proteins were broken.

Erythrocyte Membrane↗
PubMed1977

Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte.

A variant of the major transmembrane protein of the human erythrocyte has been detected following proteolytic digestion of intact erythrocytes. Pronase digestion of normal erythrocytes gives rise to a 60,000 molecular weight fragment of Protein 3, while digestion of erythrocytes with the variant protein produces two fragments of 60,000 and 63,000 molecular weight when peptides are separated by sodium dodecyl sulfate-acrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685). The two fragments cannot be resolved if electrophoresis is conducted using the continuous phosphate or Tris/acetate buffer systems. This increased molecular weight of the variant fragment does not appear to be due to increased glycosylation, since neither sialic acid residues nor terminal galactose units can be detected. Furthermore, the transmembrane segment of Protein 3 can be detected after proteolytic digestion at both the external and cytoplasmic membrane surfaces. These transmembrane segments of both the normal and the variant peptide have identical molecular weights of 20,000 to 21,000. These results suggest that the increased molecular weight of the variant peptide is due to the incorporation of an additional segment into that region of the molecule which is exposed at the cytoplasmic side of the membrane.

Electrophoresis, Disc↗
PubMed1975

The transmembrane proteins in the plasma membrane of normal human erythrocytes. Evaluation employing lactoperoxidase and proteases.

The molecular architecture of the human erythrocyte membrane has been probed using lactoperoxidase-catalyzed iodination in conjunction with Pronase hydrolysis. Resealed, hemoglobin-free ghosts were labeled at the cytoplasmic surface and the external membrane surface was subsequently digested with Pronase. Changes in size of the components labeled at the cytoplasmic surface were readily detected by sodium dodecyl sulfate gel electrophoresis. The protein 3 molecular weight class labeled at the cytoplasmic surface was extensively hydrolyzed at the external surface to produce a major 65000 molecular weight fragment and a minor 45000 molecular weight fragment. When resealed membranes were labeled on the external surface the same 65000 molecular weight labeled component is produced. These results unequivocally demonstrate that the same polypeptides in the protein 3 molecular weight class that can be labeled by lactoperoxidase at the cytoplasmic membrane surface are digested by Pronase at the external surface and are, therefore, transmembrane components. Where it is possible to label one surface of a membrane with lactoperoxidase and reseal the membrane this procedure represents an alternate method for establishing transmembrane configuration of membrane proteins.

Blood Proteins↗