have shown that the low temperature trapping of substrates in the active site of an enzyme is feasible, doubt cast by the recent work of Hassall, Johnson and Roberts ~ ~' about the mode of substrate binding proposed by Shotton 2 makcs these physiological temperature studics essential.
References
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1734 4 Atlas. D., Legit, S.. Schec/cr, I. and Berger, A. FEBS Left. 1970,
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962 l0 Sawyer, L., Shouon, D. M., Campbell, J. W., Wendell, P. L.,
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11 Tulinsky, A. and Wright, L. H. J. Mol. Biol. 1973, 81, 47 12 Bode, W. and Schwager, P. J. Mol. Biol. 1975, 98, 693 13 Huber, R., Bode, W., Kukla, D., Kohe, U. and Ryan, C. A.
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1979, 8, 299
Studies on actin fragments obtained by digestion with thrombin, BNPS-skatole and nitrothiocyanobenzoic acid Peter Johnson and Verna B. Stoekmal Department of Chemistry and Colle#e of Osteopathic Medicine, Ohio Unirersity, Athens, Ohio 45701, USA (Receiced 14 October 1981: recised 8 December 19811
1+~, hate isolat ed fi'agments of actin prepared by thrombic digestion (residues 40 374 [1], and 114 374 [II]1, BN PS-skatole cleat,age (residues 87 339 [III]i and nitrothiocyanobenzoic acid treatment (residues 10 2]6 [II/]) using preparatit:e electrophoretic aml chromatographic techniques. Analysis of the filament-lbrming and tropomyosin-hinding properties of demonstrably homo~jeneous
.li'a~lment.s recealed that only .l)'agmellt,s l and II could .lorm l:-aclin-like fihmwnts qlier otlempted remllurali+m, and thal only
fi'agment I couht hind to tropomyosin. These resuhs ill addition to our precious studies on actin.lJ'agments and chemically-modilied intact actin suqgesf that residues 1 86 and 340 374 are not required fi," F-actin.lilamenl .lbrmation, whereas residues 70 86 and/or 340 374 are essential lbr tropomyosin-binding acticity.
The actin molecule is now recognized as being one of the most widely distributed proteins in eukaryotic cell types, and considerable interest centres on its detailed molecular structure because of its apparent involvement in a variety of biological processes such as muscular contraction, protoplasmic streaming and cell division 1'2
Although the primary structure of rabbit skeletal muscle actin has been known for some time a and the sequences of other actins have recently become available 4's, relatively little is still known about the detailed tertiary structure of actins, with the latest reported X-ray crystallographic map being down to a resolution of 6 A and showing only generalized poly- peptide folding features 6. In the absence of a detailed tertiary structure for actin, chemical modification and polypeptide fragmentation studies have been used in attempts to identify the involvements of various residues and polypeptide regions in the various biological
activities of actin v t.s so that these results may be correlated with an eventual solution of the tertiary structure of actin in order to establish the location of the different types of protein, nucleotide and cation binding sites that exist on the molecule.
In earlier studies la, we reported on the renaturation and interaction characteristics of fragments of actin obtained by enzymatic digestion, and concluded that residues 1 68 of rabbit skeletal muscle actin were not essential for filament formation, t ropomyosin and myosin binding, whereas residues 208 374 were essential for these properties. In this report, we extend these findings from analysis of the properties of fragments of actin obtained by different fragmentation procedures and conclude that whereas residues i-86 and 34(>374 are not required for filament formation, residues 7(> 86 and/or 340 374 are essential for tropomyosin-binding activity.
Experimental
Actin and tropomyosin were prepared from rabbit skeletal muscle as described elsewhere 10,17. The commercially-obtained reagents used in digestion studies were thrombin (Grade 1, Sigma Chemical Co.), BNPS- skatole* [Pierce Chemical Co.) and NTCB (Eastman Chemicals).
Thrombic digestion of actin was performed according to the protocol of Muszbek et al. is except that the concentration of G-actin in the digestion was 2 mg ml t, 10 NIH units of thrombin were used per mg of actin, and the digestion was performed for 48 h at 2Y'C. After termination of the digestion by incubation for 1 h at 23 C in the presence of I mM DFP, the solution was lyophilized and then redissolved at a concentration of 10 nag ml 1 in 6 M urea, 2?; SDS, 1% /#mercaptoethanol, 50 mM phos- phate, pH 7.0. Samples (40 mg actin) were then subjected to preparative-scale electrophoresis using the apparatus of Koziarz eta/. 19 using a 5.9 x 12 cm column containing a 7.8'!; acrylamide, 0.2°; bisacrylamide gel with a running buffer of 0.2% SDS, 2 mM EDTA, 20 mM sodium acetate, 20 mM thioglycollic acid, 40 mM Tris acetate, pH 9.0. Elution of undigested actin and actin fragments was
* Abbreviations: BN PS-skatole, 2-{2-nitrophenylsulphenyl)-3- methyl-3-bromoindolenine: DFP, diisopropyl fluorophosphate; EDTA, ethylenediamine tetraacetic acid: M,, molecular weight ratio; NTCB, 2- nitro-5-thiocyanobenzoic acid: SDS, sodium dodecyl sulphate; SDS- PAGE, electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate.
monitored by analytical-scale SDS-PAGE of the collected fractions (5 ml), and fractions containing homogeneous fragments were pooled and SDS was removed by treatment with cetyl t r imethylammonium bromide 2°, after which the solutions were dialysed extensively against water and lyophilized.
Digestion of carboxymethylated actin by BNPS- skatole was performed as described previously 2~ and, after the removal of excess reagents by extraction with ethyl acetate, the aqueous solution was lyophilized,
o/ redissolved in 8 M urea, 10~,, acenc acid at a concentration of 12 mg ml- ~ and fractionated on a 2 x 200 cm column of Sephadex G-200 equilibrated in the same solvent. Fractions (2 ml) containing homogeneous actin fragments were identified by analytical-scale SDS-PAG E and were pooled, dialysed against water and lyophilized.
Digestion of actin (1 mg ml t) with NTCB was performed by the procedure of Jacobson et al. 22 and, after dialysis against 50"/o and then 51}/,i acetic acid, the solution was lyophilized and redissolved at 10 mg m1-1 in 6 M urea, 50~,] formic acid. Fractionation of samples (40 mg actin) of this solution was then performed on a 1.5 × 200 cm Sephadex G-75 column equilibrated in 10'~',g formic acid, and fractions (2 ml) containing actin fragments were identified, pooled, dialysed and lyophilized as described above.
In attempts to obtain filament formation from samples of purified actin fragments, lyophilized fragments were dissolved at 1 mg ml -~ in 4 M urea, 0.1 M [4- mercaptoethanol, 10 mM Tris HC1, pH 9.0 and then treated according to the general procedure of Mihashi 23 as described previously J4. Electron microscopic analysis and tropomyosin-binding assays on fragment preparations which had undergone the renaturation procedure were also performed according to earlier methods TM. Amino acid analyses of fragments were per- formed on 24 h acid hydrolysates ~4 using a Beckman I I9CL amino acid analyser.
Results
PuriJlcation of thrombic.[i'aqments of actin The thrombic digest was found to contain unfrag-
mented actin and two large polypeptides (M, 37 000 and
Note.s to the Editor
27000) in addition to smaller fragments (Fi~lure 1). Because it was not possible to separate these polypeptides from each other by column chromatographic procedures, preparative-scale SDS-PAGE was used and Fi.qure 1 shows the polypeptide distribution obtained in collected fractions and the pooling of tubes made for the two large fragments. From the known specificity of thrombin on actin ~8, the larger fragment ( M 37000) consists of residues 40 374 and the other fragment (M, 27000) consists of residues 114 374. These residue assignments were also confirmed by amino acid analysis of the fragments (results not shown).
Pur(ficatiotl ~?1" the BN PS-skatole l?aftmem ~f actiJl Cleavage of actin by BNPS skatole resulted in
formation of a large fragment (M~ 30000) in addition to smaller polypeptides and the presence of uncleared actin (Fiqure 2). Purification of the fragment was achieved by
~,3 • 30
0.2 [ ~
40 80 120 160 200
Tube no
Figure 2 Purification of an actin fragment from a BNPS- skatole digest. The Figure shows the absorbance profile at 280 nm of the digest on fractionation on Sephadex G-200. The inset shows densitometric scans of samples of the unfractior, ated digest (A) and the tubes pooled as indicated by the bar line in the major figure (B) which had been analysed by SDS-PAGE. The arrows indicate the approximate M r values for the polypeptides in thousands. Details of the digestion, fractionation and gel analysis are described in the Experimental section
Figure I Purification ofactin fragments from a thrombic digest. The Figure shows the polypeptide compositions of the unfractionated digest (A) and the polypeptide compositions of fractions collected from the preparative-scale SDS-PAGE apparatus. Tubes 42 47 were pooled to obtain the purified fragment of M, = 27 000 (27 t and tubes 54 56 were pooled to obtain the fragment of M - 37 000 (37 I. Thc polypeptide of Mr-43000 (43) is uncleared actin. Conditions of digestion, fractionation, and gel analysis are described in the Experimental section
Int. J. Biol. Macromol., 1982, Vol 4, June 253
Notes to the Editor
0.8
0.6 - -
g '~ 0 4 - -
0.2 - -
4O
_1 1 L _ _
80 120 160
Tube no.
Figure 3 Purification of an actin fragment from an NTCB digest. The Figure shows the absorbance profile at 280 nm of the digest on fractionation on Sephadex G-75. The inset shows densitometric scans of samples of the unfractionated digest (A) and the tubes pooled as indicated by the bar line in the major figure (B) which had been analysed by SDS-PAGE. The arrows indicate the approximate M r values for the polypeptides in thousands. Details of the digestion, fractionation and gel analysis are given in the Experimental section
chromatography of the digest on a Sephadex G-200 column and Figure 2 shows the elution profile of this column. It has previously been established 2~ that this fragment is composed of residues 87 339.
Purification of the N TCB fragment of actin Cleavage of actin with NTCB resulted in complete
degradation of the actin to a large fragment (M r 25 000) and some smaller fragments (Figure 3). The large fragment was obtained in pure form by chromatography of the digest on a Sephadex G-75 column as shown in Figure 3. Based on the known distribution of sulphydryl residues in actin and the amino acid analysis (not shown) of the fragment, this fragment comprises residues 10 216 of the actin sequence.
Filament formation by actin.~agments When the purified fragments were subjected to the
renaturation procedure described in the Experimental section and examined by electron microscopy, it was found that only the larger thrombic fragment (residues 40-374) and the BNPS-skatole fragment (residues 87 339) produced filamentous structures that were indicative of regular actin-actin interactions (Figure 4). The smaller thrombic fragment (residues 114 374) and the NTCB fragment (residues 10-216) did not produce filamentous structures.
Tropomyosin bindin 9 studies In standard tropomyosin-binding assays using actin
fragments which had undergone the renaturation procedure, only the large thrombic fragment (residues 40- 374) was found to interact with tropomyosin as evidenced by the cosedimentation of tropomyosin with filaments of this fragment. Although the other fragments were pelleted in the assay, none of them caused cosedimentation of tropomyosin, indicating the absence of tropomyosin- binding ability.
a
Figure 4 Electron micrographs of filaments obtained from renatured actin fragments. Samples were renatured and analysed by electron microscopy as described in Experimental. The scale bar represents 100 nm. (a) Thrombic fragment (M,=37000); (b) BNPS-skatole fragment; (c) uncleared actin taken through the renaturation procedure
254 Int. J. Biol. Macromol., 1982, Vol 4, June
Table I Summary of the interaction properties of actin fragments
Residues Actin in actin Cleavage Actin actin tropomyosin fragment" reagent b interaction interaction
40 374 Thrombin Yes Yes
68 374 Chymo- Yes Yes trypsin
69 374 Trypsin Yes Yes
114 374 Thrombin No No
1 207 S. aureu,s No No V8 proteasc
10 216 NTCB No No
87 339 BNPS-skatole Yes No
Based on the amino acid residue numbering for rabbit skeletal muscle actin 3 Details of the preparation of the thrombic, BNPS-skatole and NTCB fragments are given in Experimental, and the preparation and properties of the other fragments are described in Ref 14
Discussion
Previous studies ~'* have shown that it is possible to ob ta in actin f ragments (residues 69-374 and 68-374) which can form f i lamentous structures indicat ive of specific act in- actin interact ions that are similar to those in the F-ac t in f i lament and which can also interact with t r opomyos in and myosin. In extension of these data , and as summar ized in Tab le 1, the current studies present further evidence that the immedia te N- te rmina l actin sequence is not necessary for f i lament format ion or t r opomyos in - b inding as the actin f ragment of residues 40-374 p roduced by t h rombic digest ion possesses both of these propert ies . In this context , it is interest ing to note that the N- te rmina l region of actin molecules shows the greatest sequence var iabi l i ty 5, which also suggests that this region might be relat ively u n i m p o r t a n t for some of the proper t ies of actin, including nucleot ide b inding t2.
The current studies on the smaller t h rombic f ragment of actin (residues 1 14~374) and the previous work on the t rypt ic f ragment (residues 69 374) suggest that the . sequence region from residues 69 to 113 must conta in crit ical residues for f i lament format ion as the th rombic f ragment cannot be reassembled into filaments. The abi l i ty of the BNPS-ska to l e f ragment (residues 87-339) to form fi laments also suggests that the crit ical residues are, in fact, between posi t ions 87 and 113. This par t icu la r region conta ins a residue (Arg 95) that has previously been shown t3 to be involved in t r opomyos in binding, and it therefore appears that this region of the actin molecule might have two biological functions.
The results from the studies on the BNPS-ska to l e f ragment also show that an extensive C- te rmina l region (residues 340-374) of the actin molecule is not necessary for f i lament format ion, a l though the proper t ies of the N T C B fragment and previous studies 14 on the S t a p h y l o c o c c u s a u r e u s V8 protease f ragment show that the sequence region from residue 208 to 339 is necessary for this p roper ty (see Table 1). As the t rypt ic f ragment can
N o t e s to t he E d i t o r
form fi laments and interact with t ropomyos in , whereas the shor ter BNPS-ska to l e fragment forms fi laments but does not interact with t ropomyos in , it appears that one of the regions that is necessary for the t ropomyos in binding p roper ty is either residues 70-86 or 340 374. It is also possible that both of these regions are essential for t ropomyos in binding, as Cys 373 has a l ready been implicated in t ropomyos in -b ind ing 24 and residues 7(~ 86 are cont iguous with a region (residues 87 113) also implicated in t ropomyos in binding in these and earl ier studies t 3.
Tha t N- te rmina l and C- terminal regions of the actin sequence can be removed by proteolyt ic digest ions of the native molecule and that por t ions of these regions are not needed for some of the biological proper t ies of actin ~2'25 suggest that these terminal regions are not folded into the interior of the molecule, but exist as surface structures. The precise locat ions of these interest ing regions of the actin po lypept ide and the relative d isp lacement of binding sites on the two domains of actin must, however, await further progress in the solut ion of ter t iary s t ructure by X- ray crys ta l lography.
Acknowledgements
This research was carr ied out under a grant to P.J. from the Muscular Dys t rophy Associat ion. We thank Dr R. S. Hik ida for use of the electron microscope facilities.
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