DIFFERENTIAL REGULATION OF SKELETAL-MUSCLE MYOSIN-II AND BRUSH-BORDERMYOSIN-I ENZYMOLOGY AND MECHANOCHEMISTRY BY BACTERIALLY PRODUCED TROPOMYOSIN ISOFORMS

In this report, we have compared the physical properties and actin-bin ding characteristics of several bacterially produced nonmuscle and str iated muscle tropomyosins, and we have examined the effects of these i soforms on the interactions of actin with two structurally distinct cl asses of myosin: striated muscle myosin-II and brush border (BB) myosi n-I. All of the bacterially produced nonmuscle tropomyosins bind to F- actin with the expected stoichiometry and with affinities comparable t o that of a tissue produced alpha-tropomyosin, although the striated m uscle tropomyosin CTm7 has a lower affinity for F-actin than a tissue- purified striated muscle a tropomyosin. The bacterially produced isofo rms also protect F-actin from severing by villin as effectively as tis sue-purified striated muscle alpha-tropomyosin. The bacterially produc ed 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 am ino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyo sins (CTm47 and CTm74) all inhibit the actin-activated MgATPase activi ty of muscle myosin S1 by similar to 70-85%, comparable to the inhibit ion seen with tissue-purified striated muscle or tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin-activated MgATPase ac tivity of muscle myosin S1 approximately two- to threefold. The in vit ro sliding of actin filaments translocated by muscle myosin-II (2.4 mu m/sec at 19 degrees C, 5.0 mu m/s at 24 degrees C) increased 25-65% i n the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-II motility. The actin-activated MgATPa se activity of BB myosin-I was inhibited 75-90% by all of the tropomyo sin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin-I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacter ially produced tropomyosins can differentially regulate myosin enzymol ogy and mechanochemistry, and suggest a role for tropomyosin in the co ordinated regulation of myosin isoforms in vivo. (C) 1994 Wiley-Liss, Inc.