Table 1.

Characteristics of the mutant ACTs with di- or tripeptide and cysteine peptide inserts

ProteinaInsertb% Activity vs wild type
Invasivec,dHemolyticc
Di- or tripeptide inserts
 ACT wild typeNone100 ± 11100 ± 15
 ACT108/VHGly107 VHHis108 98 ± 12101 ± 14
 ACT133/VYTMet132 VYTAsp134 96 ± 1097 ± 15
 ACT233/VHGly232 VHLeu233 101 ± 13102 ± 13
 ACT336/VHGly335 VHGln336 97 ± 1198 ± 9
 ACT424/VYTMet423 VYTAla425 99 ± 1297 ± 12
 ACTΔ510–515/VYTGlu509 VYTGlu516 75 ± 9230 ± 45
 ACT524/VHArg523 VHGly524 60 ± 860 ± 9
 ACT529/VYTTrp528 VYTGly530 65 ± 875 ± 9
 ACT594/CT* Ala593 CTArg594 98 ± 1396 ± 14
 ACT607/VYTSer606 VYTGly608 96 ± 1199 ± 12
 ACTΔ615–655/VYTLeu614 VYTGln656 <1<2
 ACTΔ663–688/VYTThr662 VYTSer689 0 0
 ACT722/VYTLeu721 VYTAsn723 29 ± 423 ± 4
 ACT751/VYTLeu750 VYTAsn752 101 ± 1397 ± 14
 ACT794/VYTAsn793 VYTAsp795 37 ± 638 ± 6
 ACT890/VYTLeu889 VYTLys891 96 ± 1299 ± 14
 ACT926/CT* Ser925 CTArg926 102 ± 1495 ± 13
 ACT1166/VHGly1165 VHArg1166 69 ± 752 ± 7
 ACTΔ1245–1273/VYTSer1244 VYTGln1274 27 ± 422 ± 3
 ACT1281/VHGly1280 VHGln1281 42 ± 637 ± 5
 ACT1334/VHGly1333 VHGln1334 100 ± 1296 ± 14
 ACTΔ1387–1407/VYTGlu1386 VYTAsp1408 93 ± 1398 ± 13
 ACT1387/VYTGlu1386 VYTGly1388 100 ± 1497 ± 11
 ACT1416/VYTGln1415 VYTAsn1417 79 ± 978 ± 10
 ACT1548/VHGly1547 VHLeu1548 71 ± 965 ± 8
 ACT1623/VYTPhe1622 VYTArg1624 88 ± 791 ± 7
 ACT1648/VHArg1647 VHAsp1648 99 ± 1492 ± 9
Cysteine peptide inserts
 ACT108/E2-Cys* VRCIVH 100 ± 12100 ± 15
 ACT133/E3-Cys* VYCRILQYT 96 ± 13101 ± 13
 ACT233/E2-Cys VRCIVH 98 ± 12104 ± 15
 ACT336/E2-Cys VRCIVH NDe 99 ± 14
 ACT424/E3-Cys* VYCRILQYT 101 ± 1496 ± 12
 ACTΔ510–515/E3-Cys* VYCRILQYT <2280 ± 53
 ACT524/E2-Cys VRCIVH 0 0
 ACT529/E3-Cys* VYCRILQYT 20 ± 325 ± 4
 ACT607/E3-Cys* VYCRILQYT 88 ± 993 ± 10
 ACTΔ615–655/E3-Cys VYCRILQYT <1<2
 ACTΔ663–688/E3-Cys VYCRILQYT 0 0
 ACT722/E3-Cys* VYCRILQYT 32 ± 627 ± 4
 ACT751/E3-Cys VYCRILQYT 101 ± 1398 ± 14
 ACT794/E3-Cys* VYCRILQYT 27 ± 4.330 ± 4.8
 ACT890/E3-Cys VYCRILQYT 0 0
 ACT1166/E2-Cys VRCIVH 61 ± 750 ± 8
 ACTΔ1245–1273/E3-Cys* VYCRILQYT 26 ± 424 ± 5
 ACT1281/E2-Cys VRCIVH 28 ± 526 ± 4
 ACT1334/E2-Cys* VRCIVH 88 ± 1094 ± 14
 ACTΔ1387–1407/E3-Cys* VYCRILQYT 88 ± 795 ± 13
 ACT1387/E3-Cys* VYCRILQYT 97 ± 1296 ± 15
 ACT1416/E3-Cys* VYCRILQYT 81 ± 1075 ± 9
 ACT1548/E2-Cys VRCIVH 57 ± 660 ± 7
 ACT1623/E3-Cys VYCRILQYT 0 0
 ACT1648/E2-Cys* VRCIVH 98 ± 1396 ± 14
  • a The mutant ACT variants for which the toxin activities were determined with both the urea extracts and purified protein preparations are indicated by an asterisk. Δ indicates a deletion encompassing the indicated residues.

  • b Boldface characters indicate inserted amino acid residues at given positions in ACT. The inserted unique cysteine residues are indicated in italics.

  • c The activities are expressed as percentages of wild-type ACT activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with three different toxin preparations (n = 6).

  • d Invasive activity was determined as the AC activity translocated into sheep erythrocytes and protected against digestion by extracellularly added trypsin (5).

  • e ND, not determined.