Dear sir, Thank you for your kind letter of December 11, 2003. We are happy to know that o

Dear sir,

Thank you for your kind letter of December 11, 2003. We are happy to know that our paper entitled "Purification of Clostridium perfringens phospholipas C (@-toxin) by affinity chromatography on agarose-liked egg-yolk lipoprotein" (Art no. RPP-1265) will be acceptable. We have tried to shorten and revise the manuscript. in line with the suggestion made by one of the reviewers and yourself. I am enclosing duplicate copies of the revised version. In answer to the questions raised by the reviewer, we must admit that we have not tested for lipase activity known to exist in culture of this organism. However, since our purified phospholipase C was found homogeneous by various criteria, we believe contamination with lipase, if there is any, is likely to be slight. In any case, we will test for lipase activity as soon as possible.

As to the second question, we realized that the lipoprotein in affinity adsorbent was attacked by the enzyme to a small extent; a minute amount of phosphorycholine was always detected in the break-through peak. As reported, however, the same column can be repeatedly (at least four times) without losing its affinity for the enzyme.

We still do not know whether or not all of the multiple forms of enzyme are artifacts formed exclusively during isoeletric focusing. But some evidence is now available for the presence of at least two forms of enzyme, which are separable by methods other than isoeletric focusing, i. e. CM--or DEAE--Sephadex chromatography (unpublished data). The clarification of this question is now in progress in our laboratory.

We have accepted all the suggestions penciled in by the reviewer on the original manuscript; the abbreviation SDC has been avoided. Through these revision we have succeeded in shortening the manuscript. by two pages in total, although the page numbers have been kept unchanged. In the revised manuscript. we have put the reference numbers in the right order as requested. We would like, however, to keep the designation of the figures as it was, since we believe the suggestion may be based on a misunderstanding on the part of reviewers. As suggested, we have improved the description of the essential step involved in preparation of the affinity adsorbed (page 6,5th line from the bottom of the original manuscript) as follows; "by centrifuging the mixture at 13,000g for 15 min. to discard the precipitate." I hope that these revisions and the shortened text are satisfactory and that the revised version will be acceptable for publication in Biochimica et Biophysica Acta. I also hope the revised manuscript. will reach you before January 1.

Sincerely yours,

Nobuo Ogata, M.D.

The author wants his paper ______.

A．to be revised

B．to get a prize

C．to be published

D．to be looked through