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All rights reservedNo part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher v VI PREFACE TO PART A whose work fell into this category, we extend our apologies and request their understanding of our attempts to produce a working text, rather than to review the historical development of various concepts and techniques.This volume is a companion to Part B, Reactions and Synthesis. Part B emphasizes the synthetically important organic reactions. It adds some further mechanistic information, but is aimed primarily at introducing to students the available repertoire of organic reactions and providing an opportunity to develop a background in synthetic methodology.We believe that Parts A and B used sequentially as texts in a one-year advanced course will prepare the student to turn to the extensive review and monograph literature and to the primary journals with a thorough exposure to both the mechanistic and synthetic areas of organic chemistry.

Class III fi-tubulin, isolated from adult bovine brain, is resolved into at least seven charge variants on isoelectric focusing gels. To identify the posttranslational modifications responsible for this heterogeneity, a mixture of brain tubulins was treated with cyanogen bromide and the C-terminal fragments from the class III I3-tubulin isoforms were then isolated by binding them to the monoclonal antibody TuJ1. Combined use of tandem mass spectrometry and both subtractive and automated Edman degradation chemistry on the isolated peptides indicates that many of the isoforms differ by phosphorylation at Ser-444 plus attachment of one to six glutamic acid molecules to the side chain of the rst glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-AspAsp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys.Microtubules, assembled from two similar 50-kDa proteins designated a-and f3-tubulin, are involved in a number of important biological processes including segregation of chromosomes during cell division, cell motility, organelle transport, and maintenance of cell shape (1). Brain tubulin exhibits a high degree of heterogeneity. Up to 21 charge variants have been observed by isoelectric focusing (IEF) techniques (2-4).Both a-and 83-tubulins are encoded by small multigene families (5). Since the number of charge variants exceeds the number of tubulin genes by approximately a factor of 2, the remaining isoforms are assumed to result from posttranslational modifications. a-Tubulin is reported to undergo acetylation (6-8), glutamylation (9), and the addition and removal of a C-terminal tyrosine residue (10, 11). Phosphorylation of a single seine residue in one of the five P-tubulin isotypes expressed in brain has also been described (12-16).Class III p8-tubulin, a vertebrate isotype found only in neurons and cells in the mammalian testis (17), undergoes a developmentally regulated increase in heterogeneity in the former but not the latter tissue (18,19). The testis and earliest embryonic rat brain isoforms cofocus on an IEF gel (19). At least six additional isoforms of neuron-specific class III ,B-tubulin are detected in the adult rat or bovine brain.Previous work localized the site of heterogeneity to the C-terminal 20 amino acids (19), a highly acidic region of the protein thought to be involved in the interaction of microtubules with both calcium ions (20) and microtubule-associated proteins (21-23).To further characterize both the nature and location of structural modifications responsible for the observed heterogeneity, class III /3-tubulin from bovine brain was subjected to amino acid sequence analysis by tandem mass spectrometry. We report that the neuron-specific class III /3-tubulin isoforms result in part from phosphorylation at Ser-444 and attachment of at least three and perhaps as many as six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-GluAsp-Asp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys (YED-DEEESEAQGPK). MATERIALS AND METHODSIsolati...

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