Abstract
Two closely related isoforms of acyl carrier protein (I and II) have been purified from spinach leaves. Differences in the N-terminal amino acid sequence and amino acid composition indicate that these proteins are coded by different genes. The two spinach leaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis. Both isoforms are effectively bound by antibodies raised to acyl carrier protein I. However, in competition experiments isoform II is only about 40% effective in blocking isoform I binding to antibody. Therefore, the isoforms are immunologically related but hold only some antigenic sites in common. Immunoblot analysis ("Western blotting") of crude spinach leaf tissue extracts probed with antibody to acyl carrier protein I reveals both isoforms. In addition, both forms of acyl carrier protein are present in dark-grown leaf tissue and in isolated chloroplasts. However, in spinach seeds and roots only acyl carrier protein II can be detected. Similar results are observed with extracts of castor oil plant leaf and seed. Therefore, the expression of the two acyl carrier protein isoforms is tissue specific.
Highlights
Two closely related isoforms of acyl carrier protein (I and 11) have been purified from spinach leaves
In thepresent study we report on the separation and characterizationof two isoforms of spinach Acyl carrier protein (ACP).In addition, we present data which indicate that these two forms are coded by different genes and that theexpression of the isoforms is tissue specific
Earlier data [5] suggested the possibility that at least two species of ACP exist in spinach, the low abundance (
Summary
Vol 260, No 13, Issue of July 5, pp. 8032-8037,1985 Printed in U.S.A. Plants Have Isoformsfor Acyl CarrierProtein That Are Expressed Differently in Different Tissues*. The two spinachleaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis Both isoformsare effectivelybound by antibodies raised to acyl carrier protein I. The abbreviations used are: ACP,acyl carrier protein; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS,phosphatebuffered saline; MES, 2-(N-morpholino)ethanesulfonicacid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; MOPS, 4-morpholinepropanesulfonic acid. In thepresent study we report on the separation and characterizationof two isoforms of spinach ACP.In addition, we present data which indicate that these two forms are coded by different genes and that theexpression of the isoforms is tissue specific While this manuscript was in preparation H0j and Svendsen [11] published N-terminal amino acid sequence data indicating barley has two genes for ACP. The HPLC fractions were assayed for ACP with E. coli acyl-ACP synthetase [19]
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