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PubMed1977

Enzymatic solubilization of membrane immunoglobulin (M-Ig) from rabbit lymphocytes with phospholipase A2 (PL-A2) and phospholipase C (PL-C).

Enzymatic solubilization of M-Ig from rabbit lymph node cells was investigated using highly purified PL-A2 and PL-C. The combined treatment with PL-A2 (50 I.U./ml) and PL-C (20 I.U./ml) caused optimal solubilization of the membrane components from the 125I-labeled lymphocytes, but the treatment with either enzyme alone did not. The solubilized M-Ig was isolated and characterized as the membrane component which was specifically co-precipitable with homologous antigen-antibody complex. The solubilized M-Ig associated with some other membrane constituents was eluted in a void volume fraction by gelfiltration on a column of Sepharose 6B, and recovered at the interface between 30 and 40% sucrose layers by the density gradient centrifugation. The isolated component could be further separated by SDS-polyacrylamido gel electrophoresis into four radioactive polypeptides with apparent mol wt of 7 approximately 8 X 10(4), 4 approximately 5 X 10(4), 3 approximately 3.5 X 10(4) and 2 approximately 2.5 X 10(4), respectively. The results suggest that the enzymatic solubilization of lymphocyte M-Ig is a useful procedure to investigate further characteristics of M-Ig and their biological function related to the intracellular mechanisms of immune response.

Animals↗
PubMed1993

Transglutaminase-catalyzed incorporation of polyamines into phospholipase A2.

We have previously demonstrated that when phospholipase A2 is treated with either tissue transglutaminase or human plasma Factor XIIIa, a striking increase of its catalytic activity is observed, due to the formation of an intramolecular epsilon-(gamma-glutamyl)-lysine crosslink [Cordella-Miele et al. (1990) J. Biol. Chem. 265, 17180-17188]. Here, we report the effect of transglutaminase substrates such as mono-, di-, and polyamines on this transglutaminase-catalyzed post-translational modification of phospholipase A2. Incorporation of radioactively labeled polyamines into phospholipase A2 was demonstrated by using porcine pancreatic phospholipase A2 as a substrate in a conventional transglutaminase assay. These results were further confirmed by SDS-polyacrylamide gel electrophoresis followed by fluorography. Additionally, gamma-glutamyl-polyamine was detected and unequivocally identified in proteolytic digests of polyaminated phospholipase A2. When phospholipase A2 was incubated with transglutaminase in the presence of putrescine, spermine, spermidine, dansylcadaverine, or methylamine, a 2-3-fold increase in phospholipase A2 activity was observed. The increase of phospholipase A2 activity was found to be dependent upon the concentration of phospholipase A2, preincubation time, and the duration of the reaction. Increase in phospholipase A2 activity after transglutaminase treatment in the presence of polyamines was demonstrated using two different assay systems. Kinetic studies on phospholipase A2 pretreated with spermidine and transglutaminase demonstrated a significant increase of the apparent Vmax but no significant change in the apparent Km. Unlike phospholipase A2 pretreated with transglutaminase alone, polyaminated phospholipase A2 does not undergo non-covalent dimerization in solution. Polyaminated phospholipase A2 was further purified by chromatofocusing and was found to contain N-mono(gamma-glutamyl)-spermidine in a molar ratio of about 1:1 to phospholipase A2. Freshly purified, polyaminated phospholipase A2 had a specific activity approximately 3-fold higher than that of control phospholipase A2 treated in an identical way except for the absence of transglutaminase. To our knowledge, this is the first demonstration that transglutaminase catalyzes the incorporation of amines into a phospholipase, and that this post-translational modification increases phospholipase A2 activity.

Animals↗