Purification of the Ca2+-and Mg2+-requiring ATPase from rat brain synaptic plasma membrane

A Ca2+-ATPase (Ca2+- and Mg2+-requiring ATPase) was purified from a synaptic plasma-membrane fraction of rat brain. This enzyme had properties similar to those of plasma-membrane Ca2+-ATPases from other organs: its splitting of ATP was dependent on both Ca2+ and Mg2+, it bound in a Ca2+-dependent fashion to calmodulin-Sepharose and it cross-reacted with specific antibodies raised against human erythrocyte-membrane Ca2+-ATPase. It had an apparent Mr of 138 000, similar to those of plasma-membrane ATPases from human erythrocyte and from dog heart sarcolemma. Previous high-Ca2+-affinity ATPases observed in brain had Mr 100 000; in at least one case, such an ATPase probably represented a different type of enzyme, derived from coated vesicles.

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Purification of the NF2 Tumor Suppressor Protein from Human Erythrocytes

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/s0317167100005357 ◽

2006 ◽

Vol 33 (4) ◽

pp. 394-402 ◽

Cited By ~ 2

Author(s):

Hitesh K. Jindal ◽

Kazumi Yoshinaga ◽

Pil-Soo Seo ◽

Mohini Lutchman ◽

Patrick A. Dion ◽

...

Keyword(s):

Plasma Membrane ◽

Tumor Suppressor ◽

Erythrocyte Membrane ◽

Human Erythrocyte ◽

Mammalian Cells ◽

Tumor Suppressor Protein ◽

Human Erythrocyte Membrane ◽

Protein 4.1 ◽

Erythrocyte Plasma Membrane ◽

Inside Out

Background:Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane.Methods:Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a ~70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins.Results:These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately ~41-65,000 molecules of NF2 protein per erythrocyte.Conclusion:We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

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