Monoclonal antibody to microtubule-associated STOP protein: affinity purification of neuronal STOP activity and comparison of antigen with activity in neuronal and nonneuronal cell extracts

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Abstract
Microtubules, ordinarily cold-labile structures, are made entirely resistant to cold temperature by the presence of substoichiometric amounts of STOP (stable tubule only polypeptide), a microtubule-associated protein. We have produced a monoclonal antibody which specifically recognizes a 145-kDa protein previously implicated in STOP activity in rat brain extracts. An antibody affinity column removes both the 145-kDa protein and STOP activity from solution. A urea eluate from the affinity column contains the 145-kDa protein and exhibits substantial STOP activity. We conclude the 145-kDa protein accounts for all measurable STOP activity in rat neuronal extracts. For this work, we have developed an assay of microtubule cold stability which is generally applicable to the detection of STOP activity in various tissues. Using this assay, we show STOP activity is most abundant in neuronal tissue but is detectable in all tissues tested, with the exception of heart muscle. In all tissues that we have examined, STOP activity elutes as a single peak from heparin affinity columns, and in common with brain STOP, all activity is Ca2+-calmodulin sensitive. The monoclonal antibody recognizes the 145-kDa STOP in rat neuronal extracts but reacts with no protein in active fractions from other tissue. A similar, but not identical, analogue of brain STOP thus appears to be widespread in mammalian tissues.