PubMed · 237189
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R L Anderson, A S Dahms. 1975. D-fuconate dehydratase.. https://doi.org/10.1016/0076-6879(75)42132-2
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The presence of cytosolic S-adenosylmethionine-dependent N-methyltransferase(s) activity(ies) capable of converting phosphoethanolamine into phosphocholine has been recently demonstrated in the rat brain. At least two enzymes are involved in the methylation of phosphoethanolamine to phosphocholine and these are separable by ammonium sulphate fractionation. The enzyme catalysing the last step of this methylation process is present in the 50-80% ammonium sulphate fraction. A 220-fold purified enzyme has been obtained with sequentially employed Q-Sepharose fast flow and octyl-Sepharose CL4B column chromatography. The maximum enzyme activity was at pH 9.5. The Km values for S-adenosylmethionine, the methyl donor, and phosphodimethylethanolamine, the methyl acceptor, were 125 microM and 750 microM respectively. This phosphodimethylethanolamine N-methyltransferase was found to be calcium-dependent, with a 4-fold increase in activity at 0.5 mM-CaCl2. S-Adenosylhomocysteine at 0.5 mM caused 100% inhibition of the activity. The effects of various structural analogues on the phosphodimethylethanolamine N-methyltransferase activity were also investigated and these results suggest that the enzyme is specific to the substrate. These results provide evidence for the existence of the pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain cytosol.
A method for the purification of mouse monoclonal antibodies from hybridoma culture supernatants is described. The protocol involves the use of a combination of three previously described methods for the concentration and purification of monoclonal antibodies. Firstly, hybridomas were grown in a Diacult dialysis system (Inter Med Laboratory, Denmark) to yield milligram quantities of monoclonal antibody in a culture supernatant. Monoclonal antibodies were then purified from the culture supernatant by precipitation with polyethylene glycol 6000 (PEG 6000) and finally reprecipitated using an ammonium sulphate procedure. The PEG 6000 treatment caused a density change in the ammonium sulphate immunoglobulin precipitate, and resulted in the formation of a pellicle which contained pure mouse monoclonal antibody. The protocol removed contaminating bovine serum immunoglobulin as well as other serum and cellular protein from the monoclonal antibody preparations.
The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5'-[ beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.