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Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.

The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) is composed of a glycan linked through a glucosamine residue to an inositol phospholipid that is resistant to the action of phosphatidylinositol-specific phospholipase C. Deamination cleavage of the glucosamine with nitrous acid released the inositol phospholipid which was purified by high performance liquid chromatography. Analysis by fast atom bombardment mass spectrometry with negative ion monitoring and by the complementary technique of collision-induced dissociation revealed molecular and daughter ions that indicated a plasmanylinositol with a palmitoyl group on an inositol hydroxyl. The intact membrane anchor was released from reductively methylated human erythrocyte acetylcholinesterase by proteolysis with papain or Pronase, deacylated by base hydrolysis, and purified by high performance liquid chromatography. Positive and negative ion fast atom bombardment mass spectrometry of the major products isolated by high performance liquid chromatography indicated the following structure for the complete glycoinositol phospholipid anchor. (formula; see text) Methylation of free amino groups by reduction with deuterium instead of hydrogen permitted determination of the number of free amino groups in individual fragment ions as further confirmation of structural assignments. The structure of the glycan portion of the human erythrocyte acetylcholinesterase membrane anchor appears to be similar to that described for Trypanosome brucei variant surface glycoprotein MITat 1.4 (variant 117) (Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Science 239, 753-759) except for the absence of a galactose antenna and the presence of a phosphorylethanolamine on the hexose adjacent to glucosamine.

The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) contains a novel inositol phospholipid which in this and the accompanying paper (Roberts, W.L., Santikarn, S., Reinhold, V.N., and Rosenberry, T.L. (1988) J. Biol. Chem 263, 18776-18784) is shown to be a plasmanylinositol that is palmitoylated on the inositol ring. The inositol phospholipid was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I] iodophenyl)diazirine and characterized by various chemical and enzymatic cleavage procedures whose products were analyzed by thin layer chromatography and autoradiography or gas chromatography. Acidic methanolysis of human erythrocyte acetylcholinesterase (Ehu AChE) revealed 18:0 and 18:1 alkylglycerols (0.55 and 0.20 mol/mol AChE, respectively). Acetolysis was shown by TLC to release alkylacylglycerol acetates from Ehu AChE. Analysis by gas chromatography revealed that 83% of the alkylacylglycerol acetates contained an 18:0 or 18:1 1-alkyl group and a 22:4 (n - 6), 22:5 (n - 3), or 22:6 (n - 3) 2-acyl group. The inositol phospholipid is linked to the anchor by a glucosamine in glycosidic linkage, and deamination with nitrous acid cleaved the glycosidic linkage and released the phospholipid. The deamination and acetolysis products from Ehu AChE were purified by high performance liquid chromatography, and fatty acid analysis following acidic methanolysis of the purified products revealed that 2 fatty acid residues were associated with the deamination product and only one with the alkylacylglycerol acetolysis product. The other fatty acid residue was primarily palmitate and was indicated to be in ester linkage to an inositol hydroxyl(s). This linkage was shown to be responsible for the resistance of the inositol phospholipid to cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase. Deacylation of the inositol phospholipid deamination product by treatment with base removed this palmitoyl group and facilitated release of alkyl- and alkylacylglycerol species by phosphatidylinositol-specific phospholipase C with concomitant formation of inositol 1-phosphate. In contrast, digestion of Ehu AChE with a recently reported anchor-specific phospholipase D resulted in release of plasmanic acids from the intact palmitoylated plasmanylinositol.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated “cores,” (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.