Robert Haas

Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: Application to human erythrocyte acetylcholinesterase

A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its N alpha-methyl and N alpha,N alpha-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.

Identification and analysis of glycoinositol phospholipid anchors in membrane proteins.

Publisher Summary This chapter highlights the criteria (identification and analysis) to demonstrate the presence of a glycoinositol phospholipid in a protein of interest. Some of the glycoinositol phospholipid anchors identified include trypanosome variant surface glycoproteins (VSG), decay accelerating factor (DAF), Thy-1, and G2 acetylcholinesterase (AChE). All appear to reside on the extracellular face of the cell plasma membrane. In most cases, the identification has been based on their susceptibility to release from the cell surface by purified bacterial phosphatidylinositol- specific phospholipase C (PIPLC). Cleavage of anchored proteins with exogenous PIPLC has been achieved in a variety of culture media or isotonic buffers. The loss of anchored Thy-1 antigen from the cells is monitored by flow cytometry. In all anchored proteins, the inositol phospholipid is the only nonionic detergent-binding domain. Cleavage of the anchor by PIPLC removes the hydrophobic diacylglycerol or alkylacylglycerol and generates a hydrophilic protein that retains the glycan portion of the anchor. Two procedures most widely employed to demonstrate the loss of detergent-binding capacity following incubation with PIPLC include phase partitioning with Triton X-114 and nondenaturing poly acrylamide gel electrophoresis (PAGE).

Binding of fibronectin to gelatin and heparin: effect of surface denaturation and detergents.

Fibronectin Collagen Heparan sulfate Affinity chromatography Detergent Adhesion

Substrate-Selective Inhibition and Peripheral Site Labeling of Acetylcholinesterase by Platinum(Terpyridine)Chloride

Acetylcholinesterase (AChE) catalyzes the hydrolysis of its physiological substrate acetylcholine as well as of a number of other acetic acid esters. A key feature of AChE is its speed in cleaving substrates (Rosenberry, 1975a). The second order rate constant for acetylcholine hydrolysis (kcat/Kapp = 2 × 108 M-1s-1) approaches the value expected for a diffusion-controlled reaction. The turnover rate for acetylcholine (kcat = 2 x 104 s-1) is at the upper limit of reactions catalyzed by general acid-base catalysis. Quinn (1987) has noted that an enzyme with such a high catalytic efficiency is likely to have evolved to a point where the free energies of successive transition states are nearly matched and comparable to the diffusional barrier for substrate binding.