scholarly journals Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm

1978 ◽  
Vol 176 (1) ◽  
pp. 75-82 ◽  
Author(s):  
D L Schneider ◽  
J Burnside ◽  
F R Gorga ◽  
C J Nettleton
Keyword(s):  
Membrane Proteins ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thiol Groups ◽  
Liver Lysosomes ◽  
Lysosomal Membrane Proteins ◽  
Rat Liver Lysosomes ◽  
Lysosomal Protein

Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances.

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

1988 ◽  
Vol 66 (4) ◽  
pp. 273-278 ◽  
Author(s):  
C. Anthony Rupar ◽  
Jeffery D. Whitehall
Keyword(s):  
Membrane Proteins ◽  
Rat Liver ◽  
Concanavalin A ◽  
Ricinus Communis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peanut Agglutinin ◽  
Ulex Europaeus ◽  
Ricinus Communis Agglutinin ◽  
Lysosome Membrane

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze–thaw shock and membranes were isolated. The release of β-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.

Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns

1978 ◽  
Vol 56 (7) ◽  
pp. 713-721 ◽  
Author(s):  
I. M. Yousef ◽  
R. K. Murray
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Canalicular Membrane ◽  
Polypeptide Patterns ◽  
Cell Plasma ◽  
Bile Canalicular Membrane ◽  
And Storage

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3–CaCl2, and K2HPO4–KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3–CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (> 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to actin were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris–HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3–CaCl2 appeared to be the best buffer and Tris–HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.

scholarly journals Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane

1982 ◽  
Vol 208 (1) ◽  
pp. 77-82 ◽  
Author(s):  
M Lindén ◽  
P Gellerfors ◽  
B D Nelson
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Charge Heterogeneity

A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.

scholarly journals Genistein inhibits glucose and sulphate transport in isolated rat liver lysosomes

2009 ◽  
Vol 103 (2) ◽  
pp. 197-205 ◽  
Author(s):  
Hsu-Fang Chou ◽  
Kun-Hung Chuang ◽  
Yi-Shan Tsai ◽  
Yi-Ju Chen
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Rat Liver ◽  
Uptake Kinetics ◽  
Isolated Rat Liver ◽  
Sulphate Transport ◽  
Genistein Treatment ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Isolated Rat

Genistein and daidzein are known to have both beneficial and adverse effects on human health due to their many biological actions at the cellular level. Both isoflavones have been shown to inhibit GLUT-mediated glucose transport across the plasma membrane of mammalian cells. Since lysosomal membrane transport is essential for maintaining cellular homeostasis, the present study examined the effects of genistein and daidzein on glucose and sulphate transport in isolated rat liver lysosomes. Both genistein and daidzein significantly inhibited lysosomal glucose uptake. Genistein was a more potent glucose transport inhibitor than daidzein, with a half-maximum inhibitory concentration (IC50) of 45 μmol/l compared with 71 μmol/l for daidzein. Uptake kinetics of d-glucose showed a significant decrease in Vmax (control:genistein treat = 1489 (sem 91):507 (sem 76) pmol/unit of β-hexosaminidase per 15 s) without a change in Km. The presence of 50 μm-genistein in the medium also reduced glucose efflux from lysosomes preloaded with 100 mm-d-glucose. Genistein also inhibited lysosomal sulphate transport. Similar to its effects on glucose uptake kinetics, genistein treatment caused a significant decrease in sulphate uptake Vmax (control:genistein treat = 87 (sem 4):59 (sem 5) pmol/unit of β-hexosaminidase per 30 s), while the Km was not affected. The evidence provided by the present study suggests that the most likely mechanism of lysosomal glucose transport inhibition by genistein is via direct interaction between genistein and the transporter, rather than mediation by tyrosine kinase inactivation. Genistein likely has a similar mechanism of directly inhibiting sulphate transporter.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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