The characterization of invertebrate troponin C

0305-0491/84 $3.00 + 0.00 © 1984 Pergamon Press Ltd

4, pp. 525-529, 1984

~ACTERIZATION OF IN TROPONIN C SHIMA, TAKAHIDE TSUCHIYA, WILLIA and JUICHIRO J. MATSUMOTO L~epartment Ol t..nemlstry, Faculty faculty oI of Science and ano Technology, lec] S Chiyoda-ku, Tokyo 102, Japan; and *Department of Physiology, Boston University, School Schoo of Medici

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ity, 7-1, Kioi-cho, [A 02118, USA

(Received 23 March 1984) Abstraet--l. TNCs from lobster, mussel, and squid migral rated with ral of 18,000. 2. Electrophoretic mobilities in the presence or absence of Ca 2+ w mobility of rabbit TNC was greater in the presence of Ca :+ than it its tested migrated identically, whether Ca :+ was present or not. r 3. The Ca2÷-binding capacity of invertebrate TNCs was only one 4. The ~t-helix contents in the presence or absence of Ca C~ 2+ were cc a value of 16% and invertebrate TNCs by 4~o. 5. Antibodies to loligo TNC did not cross-react with rabbit rabb TNC, bu TNCs.

INTRODUCTION

The he contraction of vertebrate striated muscles is controlled ~ntrolled by the T M - T N system. In contrast, regulation tion of the invertebrate muscles, particularly molluscan Lscan muscle, depends on the myosin light chains (Lehman ~ehman et al., 1972, 1975). However, the occurrence ofE"T M - T N such as in the lobster, the scallop, and the ligo suggests that a thin filament-linked regulatory loli and fimc~ticm function in in ininr ~ n t,~nt :and system may also be npresent vertebrate muscles (Regenstein itein and Szent-Gyrrgyi, 1975, Goldberg and Lehman m, 1978, Tsuchiya et al., 1978, Konno, 1978). Even though two systems may exist in a single muscle in these invertebrates, the physiological function of dual-re Lual-regulation is not yet known. In the present stud' ady we investigated the Lte mus muscles by comparing T N C s of various invertebrate the properties of these T N C sS with those of vertebrate muscles. D METrIODS MATERIALS AND Rabbit (Oryctolagus cuniculus vat. dornesticus ) was :rials Center K. K., lobster obtained from Biological Materials eidiya K. K., scallop (Pati(Homarus americanus ) from Meidi' nopecten yessoensis), octopus (Octopus vulgaris), loligo (Doryteuthis bleekeri), and squidd (Tadarodespacificus)from Tokyo Central Wholesale Fisha Market. Mussel (Mytilus edulis) was collected from Tokyo Bay. The back muscle of rabbit, tail muscle of lobster, adductor muscles of scallop and mussel. mussel, and mantle muscles of loligo and squid were used inteach each preparation of TNCs. Rabbit TN was prepared by the method of Ebashi et al. (1971). Invertebrate TNs were extracted by the method of Regenstein and Szent-Gyrrgyi (1975). TNCs were purified O975). Antiby the method of Lehman and Szent-Gvrr~vi ;y bodies to loligo TNC were raised accord of Lehman et al. (1980). To ensure the used for immunization, the TNC prepan purified by the preparative electropho

n apparent mol. wt the electrophoretic invertebrate TNCs aolecule. t TNC changed by ~ith their molluscan

1980)~). Antisera were tested for reaction reactic with antigen, as described by Lehman et al. (1980). SD ;DS-PAGE was performed as desc described by Laemmli (1970~) and 6M urea alkaline PAGE was performed according to tc Head and Perry (1974). Protein concentration was measured by the method of Lowry e t aal. (1951) using BSA recast as a.,standard. Ca2÷-binding capacity vwas measured using uilibrium dialysis (Head et al., 1979) 1 and quin II equili fluorescence (Tsien, 1980; Tsien et al., 1982). The natural fluore ~resence or absence of fluorescence was measured in the pres fluore Ca 2÷ (excitation wavelength 280 nm, emission e wavelength 300 nm). Fluorescence measurement was wa carried out using Hitachi 204 Fluorescence Spectrophoto )hotometer at room temperature. Circular dichroic spectra were we recorded with a Jasco J-500 spectropolarimeter. RESULTS Characterization o f invertebrate T ~N C s S D S - P A G E patterns of T N C s se:parated from the invertebrate muscles by SP-Sephade 9hadex C-50 are shown and from in Fig. 1. T N C s from lobster,, an arthropod, artl squid, a mollusc, migrate with rabbit ra T N C at an However, although the apparent mol. wt of 18,000. H owe ubiquitous Ca 2+ binding protein, caimodulin, is in many respects similar to T N C , we w found that the they do not T N C s differ from calmodulin because bec~ 9revious investigation activate the P D E assay. In our prev (Tsuchiya et al., 1982), we reported that loligo T N C antiserum cross-reacts with loligo thin filaments; these results confirm that this protei )rotein is a component of thin filaments, presumably a TNC-like "[ protein. When electrophoresis is carried out o~ in urea under alkaline conditions (in the absence of SDS), mollus.ca_nT N C s migrate at the_ same rate; rat~ the mobility of t greater and the mobility (Fig. 2), confirming the :11 (1980). When electro~sence or absence of Ca 2+ horetic mobility of rabbit

YUKIOSHIMAet

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T N C was foun than in its abs migrated idenl not.

er in the presence of Ca :~ invertebrate T N C s tested ler Ca 2 was present or

Ca 2 ~-binding s The Ca: ' -bi by using 45Ce Rabbit T N C contrast, inver These results c Thes 1980).

:y of T N C s was measured scent reagent (Table 1). + ions per molecule; in bind only one Ca 2+ ion. of Lehman et al. (1976,

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(a) (b) (c) (d) Co) (f) (g) g. 1. SDS-PAGE of various kinds of TNCs. Gels (15°/~, were stained with Coomassie brilliant blue R. (a)I rabbit TNC, (b) lobster TNC, (c) mussel TNC, (d) dlop TNC, (e) octopus TNC, (f) loligo TNC, (g) squid scallo TNC. acr,rylamide}

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It is general] that ~-helical content of T N (C changes :ling (Nogy and Gergely, 1979 ; Burtnic t h e C D spectra of T N C s from various i~ which had been dialyzed ~mst solutic agair g either 2 m M CaC1, or 11 mR mM EGTA, a Fig. 3. he spectra of invertebrate In the preset ~roteins had about 3 5 ~ TNC7s suggest c~-hel a-helical confi mewhat lower than the Lbbit T N C . Moreover, in 45"°° -helical the aOabsence .b'3k, lll,~, Iof dl LO. ~ tll~ ~ Z --helical ll~ll~O.I content of rabbit TNC T N C increases but the increase in invertebrate ir TNCs' a-helical content is smaller. The ~:~-helical contents in ~-hel Ca -'+ (by calculating the 1presence and absence of Ca-' from [0]222) were compared: rabbit T N C changed by a val value of 16~ and invertebrate TI' T N C s by 4!~0 (Table

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(g) ~) contained C a C I 2, ie brilliant blue R. ussel TNC; (d) and g'), squid TNC.

he characterization of invertebrate tropon CaZ+-bindingof TNCs Calcium binding ~2+mole/mole TNC) 4.0 1.3 1.0 1.1 1.3 1.3 0.75

527

those of rabb structures are h nological deten T N C and stuc TNCs. Antibo( with rabbit T N TNCs. Moreox with this anti, immunological findings of Leh T N componenl c munication).

ascertain if the T N C nd possess similar immuaised antibodies to loligo ~ss-reactivity with other T N C did not cross-react zted with their molluscan 'NC, did not cross-react ,', it also had different "hese results confirm the Lowed that antibodies to l-specific (personal com-

• The respective change in s-helical content (16/4~o) ay reflect the respective difference in Ca 2+ binding /1) between vertebrate TNC.

~munodiffusion studies The physiochemical properties of invertebrate NCs are similar to one another but different from !

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;ION Table 2 sun Ta inver' invertebrate an, wt of 18,000 migr~ rate differer

ae of the properties of ~s. All TNCs have a tool. ;d by S D S - P A G E , but :ea alkaline P A G E (non-

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2OO Z22 Wav#h Fig. 3. Circular dichroic spectra TNC, (b) lobster TNC, (c) mus TNC. Proteins were scanned in (

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of Ca 2+, (a) rabbit go TNC, (g) squid ['er, pH 7.0, 50 mM

YUKIO SHIMA et al.

able 2. Comparison of between TNCs of three Mammalia Arthropo (rabbit) (lobster ight 18,000 18,000 ¢lobility* I 0.8 ?alcium sensitivity + 4 I TNC) e intensity tent (°o) *Relative

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)S). The better difference in mobility is a reflection the lower acidity between invertebrate and verteate T N C s (Lehman et al., 1980). In contrast to rtebrate T N C , invertebrate T N C s migrate at the me rate on alkaline P A G E in the presence or ,sence of Ca 2+. It is possible that this property is fluenced by the magnitude of the Ca 2+ binding, oreover, our results show smaller total o-helical ntent and fluorescence intensity of invertebrate XrC's. Furthermore, as mentioned above, some con-

formnational ch ing Ca 2+ . Considering C( :rtebrate m invez regulatory sys binding of ii myo,)sin-linked linked regulat synergistically. When comp W

Cs were induced by bindulation of contraction of formed by a dual-linked is likely that the Ca 2+ TNCs supplements the ability, and that myosinin-linked regulation act nunological properties of"

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(b) Fig. 4. Immunodiffusion reactio TNC; left hand well, loligo TNC .

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Bottom well, rabbit JC. (b) Bottom well, ell, mussel TNC.

'he characterization of invertebrate tropo~ at the antibodies to er molluscan T N C s NC, which suggests that some antigenic dering differences in ted by Ca2+-binding ces of T N C s , it is of m u c h T N C variants

REFERENCES

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