IDENTIFICATION AND CHARACTERIZATION OF AN ALPHA-ACTININ LIKE PROTEIN FROM HONEYBEE INDIRECT FLIGHT MUSCLE

Antibody was produced in rabbits to a 92,000 dalton protein from Z-discs isolated with 0.43% lactic acid from honeybee indirect flight muscle. Solubilized Z-disc proteins and whole myofibrils showed a single precipitin line in Ouchterlony immunodiffusion tests, and radioimmunostaining of gel slices and immunoperoxidase staining of Western blots showed specific antibody interaction with the 92,000 dalton protein.; Indirect immunofluorescence staining with this antibody showed specific staining of the Z-bands of honeybee indirect flight muscle, and en face views of the isolated Z-discs showed immunofluorescence staining throughout the Z-disc. The antibody also stained the Z-bands of asynchronous fibrillar flight muscle of insects of the orders Hemiptera, Coleoptera, and Diptera, as well as synchronous nonfibrillar insect muscle, but did not crossreact with vertebrate skeletal muscle Z-bands. The 92,000 dalton protein was presumptively identified as a member of the alpha-actinin family when antibody produced to vertebrate smooth muscle alpha-actinin bound to it in immunoblot experiments.; The following similarities between vertebrate, honeybee, and other insect alpha-actinins were found: (1) The honeybee alpha-actinin and alpha-actinin from rabbit skeletal myofibrils coelectrophoresed in the continuous gel system of Weber and Osborn. (2) Honeybee alpha-actinin had at least 8 isoelectric variants (pI 5.75 - 7.25), with electrophoretic patterns very similar to those of vertebrate skeletal and smooth muscle alpha-actinin. (3) The amino acid content of the honeybee 92,000 dalton protein was very similar to those of published amino acid analyses of Lethocerus and dung beetle alpha-actinin.; Honeybee alpha-actinin, however, had a higher electrophoretic mobility than alpha-actinins from chicken gizzard smooth muscle and rabbit skeletal myofibrils when run in the discontinuous gel system of Neville. These differences in electrophoretic mobility were not due to proteolytic digestion or to the presence of associated carbohydrates. Digestion with papain of alpha-actinin from honeybee flight muscle and from chicken gizzard smooth muscle resulted in sets of peptide fragments that were quite dissimilar.