A MONOCLONAL-ANTIBODY THAT DISTINGUISHES LATENT AND ACTIVE FORMS OF THE PROTEASOME (MULTICATALYTIC PROTEINASE COMPLEX)

Monoclonal antibodies (mAbs) were generated to proteasome purified fro m human erythrocytes. Five of six proteasome-specific mAbs reacted wit h three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted r eactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its acti ve state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome p reparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, lo ss of this component and generation of the 28-kDa component are not ob ligatory for activation. The 32- and 28-kDa subunits can each be furth er resolved into three components by isoelectric focusing. The apparen t loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more bassic pI for each polypeptide. Weste rn blots of the early steps of proteasome purification reveal an AP5C1 0-reactive protein at 41 kDa. This protein was separated from proteaso mes by sizing chromatography and may represent a pool of precursor sub units. Since the 32-kDa subunit appears necessary for latency, it is s peculated to play a regulatory role in ATP-dependent proteolytic activ ity.