J. Biochem. 83, 795-798 (1978)

Improved Preparation and Cooperative Calcium

Contraction of Glycerinated Vorticella1

Hiroshi ASAI, Tsutomu OCHIAI, Keiji FUKUI , Masato WATANABE, and Fumio KANO2

Department of Physics, Waseda University,

Nishi-okubo, Shinjuku-ku, Tokyo 160

Received for publication, September 9, 1977

An improved method for the preparation of glycerinated convallaria was investigated.

The pretreatment of living vorticellas with a medium containing 0.1 % saponins and subsequent

treatment with an extraction medium containing 35 % glycerol at about 0•Ž was satisfactory.

The equilibrium average length of contractile stalks of glycerinated vorticellas was measured at

various free calcium concentrations in the reaction medium. It was found that the contractile

element in the spasmoneme of the stalk is contracted by a cooperative interaction involving at

least two calcium ions.

The stalk of a peritrich Vorticella is usually times encountered a similar problem, which is 0.2-0.5mM long and can coil into a left-handed probably mainly due to disconnection of the helix. The coiling is produced by the contraction contracted spasmoneme from the sheath. Thus, of an intracytoplasmic thread called the spasmo we sought to develop an improved method for the neme, which is coiled spirally within a cylindrical preparation of glycerinated Vorticella. sheath of the stalk. Hoffmann-Berling (1) demon It was found after several trials that the strated that the stalk of glycerinated Vorticella pretreatment of living vorticellas with 0.1 coiled in the presence of calcium ions and uncoiled saponins followed by gradual glycerination is when calcium was subsequently removed by the satisfactory. This report describes this improved addition of a calcium chelater, e.g., EGTA. preparation method and the cooperative properties However, Townes and Brown (2) reported of calcium-contractile Vorticella. that the reversible contraction of glycerinated preparations by calcium ions becomes weak, MATERIALS AND METHODS especially at pH's far from pH 6.8. We some The vorticella used was a clone isolated from a 1 This work was aided in part by the Saneyoshi Science population taken from a local pond and identified Foundation. as V. convallaria. Stock cultures of the clone 2 Present address: Tokyo Metropolitan Laboratory of were maintained on cover glass fragments in hay Public Health. infusion medium previously inoculated with Aero Abbreviations: EGTA, ethyleneglycol-bis(P-amino-ethyl bactor aerogenes as prey for the vorticellas. The ether) N,N•Œ-tetraacetic acid; EDTA, ethylenediamine cover glass fragments with vorticellas attached to tetraacetic acid.

Vol. 83, No. 3, 1978 795 796 H. ASAI, T. OCHIAI, K. FUKUI, M. WATANABE, and F. KANO both surfaces were transferred from the culture medium to a medium containing 0.1 % saponins, RESULTS AND DISCUSSION 4mM EDTA, 100mM KCl, and 20mM histidine

HCl buffer (pH 6.8) at room temperature. The When living vorticellas cultured in hay infusion

medium was cooled gradually using an ice-cold medium were immersed directly in 50% glycerol, water bath and kept for 45 min at this temperature. the vorticellas were very often coiled tightly so

The gradual cooling of the medium in which the that the spasmoneme became detached from the

cover glass fragments with vorticellas were placed sheath and lost its reversible contractivility. The

was essential for obtaining good preparations. bell-shaped zooid was also easily detached from

The cover glass fragments were then immersed in the sheath. Thus, it was important to glycerinate

a new extraction medium containing 35% glycerol, the living vorticellas by means of a gradual increase

4mM EDTA, 100mM KCl, and 20mM histidine of glycerol concentration. It was also important

HCl (pH 6.8) at about 0•Ž. The extraction was to change the temperature of the extraction medium continued for 60 min at this temperature. For or reaction mixture gradually. After several

prolonged storage (up to 6 months) of glycerinated trials, it was found, however, that the glycerinated preparations of vorticellas attached to the cover models thus prepared still sometimes lost the

glass fragments, the extraction medium was ability to exhibit reversible contraction-extension replaced by a new extraction medium containing cycles if the pretreatment with 0.1 % saponins was

50 % glycerol, 4mM EDTA, 100mM KCl, and 20 omitted, and if EGTA rather than EDTA was

mm histidine-HCl buffer (pH 6.8) and stored at used as a Ca-buffer in reaction mixtures. Of

-15 -20•Ž. For immediate use of glycerinated course, the use of EGTA is essential to adjust preparations, 35 % glycerol was removed by rinsing free calcium concentration in a reaction mixture with a solution containing 4mM EDTA, 100mM containing free magnesium ions. After pretreat

KCl, and 20mM histidine-HCl (pH 6.8) at about ment of living vorticellas with 0.1 % saponins, the 0•Ž for 10 min. Then, the rinsing medium was subsequent treatment with 35 % glycerol for 60 min warmed gradually to room temperature and a was sufficient to obtain excellent models, as de cover glass fragment with attached vorticellas was scribed in the previous section. It was found, placed in a well on a slide glass containing a during the course of various experiments, that for suitable reaction mixture in the presence of calcium immediate use the treatment of living vorticellas

buffer. For the use of stored preparations of with 0.1 % saponins alone for 2 h was sufficient. glycerinated Vorticella in 50% glycerol, the glycerol Actually, Hofmann-Berling (1) stated in his paper was removed by rinsing with a solution containing that 0.1 % saponins can be used to pretreat living

4mM EDTA, 100mM KCl, and 20mM histidine Vorticella gracilis without mentioning the effects

HCl (pH 6.8) at about 0•Ž for 10 min. Then, of saponin pretreatment on the vorticellas. It is the rinsing medium was warmed gradually to room generally considered that one of the effects of sapo temperature. The subsequent procedures for ex nin treatment of a biological membrane is to en periments were similar to those already described. hance the permeability of the membrane to various All reaction mixtures for measuring the ions and also to some macromolecules (5). How stalk lengths of vorticellas contained 4mM EGTA ever, subsequent investigators of glycerinated vorti as the calcium buffer, 100mM KCl and 20mM cellas, i.e. Townes and Brown (2), Amos (4), and histidine-HCl (pH 6.8). A value of 5 x 105 m-1 others, have not followed Hofmann-Berling's pre was used as the stability constant of EGTA and treatment method. Thus, our method of preparing calcium ions to estimate pCa at pH 6.8 (3). The glycerinated vorticellas is essentially a redevelop stalk lengths of vorticellas attached to a cover ment of his method. glass fragment on the slide glass were measured The contraction and extension cycles of two by means of an ocular micrometer at a magnifica glycerinated stalks thus prepared are shown in tion 40 x or 100 x after each change of concentra Fig. 1. The cycles were induced more than 135 tion of free calcium. times by immersing the stalks alternately in reaction

solutions containing 1ƒÊM free calcium (estimated)

and 4mM EGTA. The fractional stalk length

J. Biochem. PREPARATION AND CONTRACTION OF GLYCERINATED Vorticella 797

(L) is taken as 1.0 for the fully extended state and chemical one and does not involve ATP or other

as 0.0 for the completely contracted state. As glycerol-extractable chemicals. shown by the broken line in Fig. 1, the ability to When the free calcium concentration in the

contract in 1ƒÊM free calcium gradually declined reaction mixture was changed from 10 pat to

with repeated cycles of contraction and extension. 2 [LM, the stalk extended partly, as shown im

As shown by the cross in this figure, the addition Fig. 2, and maintained its extended state for more

of 1mM ATP did not affect the contractibility of than 1 h. The time required to extend after

the stalks. The results confirm that the contrac changing the medium was less than I min. The

tion of the vorticella stalk is a purely mechano required time to contract after changing to a

medium containing a higher concentration of free calcium ions was similar. It is thus con

sidered that during contraction and extension

cycles the equilibrium stalk length is reached

within 1 min.

The average fractional stalk length of many

vorticellas is plotted against the free calcium concentration in reaction mixtures in Fig. 3. At

free calcium concentrations lower than 0.1ƒÊM,

the glycerinated stalks are fully extended. At

free calcium concentrations higher than 10ƒÊM,

the stalks are invariably contracted. The frac

tional stalk length (L) can be expressed as a func

tion of free calcium concentration by a Hill's

Fig. 1. Plot of many reversible contraction-extension equation, which is frequently applied to ligand

cycles of glycerinated . The reac binding with an allosteric protein, tion mixtures for the contracted states of one stalk (ƒ•) and another stalk (ƒ¢) contained 100mM KCl, 20mM histidine-HCI (pH 6.8), 4 mat EGTA, and 1ƒÊM (esti mated) free calcium ions. (•~) indicates the presence of 1mM ATP in the above reaction mixture. The reaction mixture for the fully extended state of the stalks (•œ) contained 100mM KCI, 20mM histidine-HCI

(pH 6.8), and 4mM EGTA.

Fig. 2. Time courses of stalk length in various free calcium concentrations.

Vol. 83, No. 3, 1978 798 H. ASAI, T. OCHIAI, K. FUKUI, M. WATANABE, and F. KANO

Fig. 3. Relationship between the normalized overall stalk length and pCa. A logarithmic plot of (1-L)/L against pCa is shown in the insert for estimation of the Hill's parameter n and affinity Km.

where [Ca] is the concentration of free calcium in the coiled-stalk length after full coiling was more ions and pCa=-log10[Ca]. Km is the free than 80 %. It is also noteworthy that the threshold calcium concentration when L becomes 0.5. free calcium concentration for the contraction of The Hill's parameter n is indicative of co a portion of the stalk near the bell-shaped body of operativity in ligand binding to an allosteric pro a zooid is somewhat lower than that near the

tein, for example, hemoglobin. As shown in the rootlet attached to the cover glass. The L value

insert to Fig. 3, n is about 2.0 and Km is about summarized in Fig. 3 is the average of a large

1.0ƒÊM. This implies that the cooperative binding number of normalized overall stalk lengths. If it

of two calcium ions to a contractile element in the is possible to investigate precisely the contractility

spasmoneme induces the contraction and that the of a portion of a stalk with increasing free calcium

free calcium concentration for half average con concentration, the Hill's parameter n should be traction of the spasmoneme is about 1.011 M. found to be larger than 2.0. Such measurements, Our result is in conflict with Amos's result (4), however, will be very difficult.

since he reported that his preparation of the We are grateful to Dr. Isamu Morishita of Ebara isolated spasmoneme of geniculatum Infillco Co. and to Dr. Ryoko Kawamura of Hiroshima appeared to contract noncooperatively with in University for their kind suggestions regarding the creasing free calcium concentration, though the culture of Vorticella. result was not conclusive. The discrepancy might be due to differences in the species used, in the REFERENCES preparation method or more probably in the accuracy of the data. In the case of the isolated 1. Hoffmann-Berling, H. (1958) Biochim. Biophys. Acta 27,247-258 spasmoneme of Zoothamnium geniculatum used 2. Townes, M.M. & Brown, D.E.S. (1965) J. Cell. Comp. by Amos, the total change in the spasmoneme Physiol. 65, 261-269 length after full contraction was about 17%. 3. Ogawa, Y. (1968) J. Biochem. 64, 255-257 Therefore, accurate estimation of cooperativity 4. Amos, W.B. (1975) Molecules and Cell Movement in the interaction of the contractile element of the (Inoue S. & Stephens, R.E., eds.) pp. 411-436, Raven Zoothamnium spasmoneme with calcium ions is Press, New York difficult. In the case of the glycerinated model of 5. Ohtsuki, I. & Ozawa, E. (1977) Cell Stru. Func.

Vorticella convallaria used by us, the total change 2 (No. 4), in press

J. Biochem.