Further observations on the native and recrystallized crystals of the amoeba Amoeba proteus

FURTHER OBSERVATIONS ON THE NATIVE AND RECRYSTALLIZED CRYSTALS OF THE AMOEBA AMOEBA D. CARLSTROM

PROTEUS and K. MAX

MBLLER

Department of Medical Physics, Karolinska Znstitutet, Stockholm, Sweden, and Curlsberg Laboratorium, Physiological Department, Copenhagen, Denmark Received February

8, 1961

G HII:I:IS’S

demonstration of the identity of recrystallized amoeba crystals to triuret [S, 71 paved the way for far greater advances in the study of the structure of native amoeba crystals than outlined in Grunbaum, Max Mc?ller, anti Thomas’s brief note [10!, but Griffin’s concept of the morphology, and cons?crystals (of triurrt) cannot 1~ quentlp the structure, of the recrystallized correct. \\‘e therefore felt compelled to expound our present ideas roncerning the relationship hetlveen the structures of the genuine and of the recrystallized hlax Mvrller’s, and ‘Thomas’s final amoeba crystals, although Grunbaum’s, publirations ;9, 11, 11, 161 have not yet been printed. This w-evaluation of’ the studies mentioned is mainly due to the S-ray crystallographical \\ork carried out hy one of the present authors (I). C). MATERIALS

(a) Genuine amoeba crystals1 had been isolated from A. poterrs as dewrihed by Grunbaum [9]; about 100 pug was available. (1)) Recrystallized amoeba crystals1 had been prepared hy shaking about 1.50 /lg isolated, genuine amoeba crystals with 200 ,~l jvater at SO”C and keeping the mixture for about forty-eight hours at room temperature. I)uring all of this time crystals were present. Microscopy shelved that all genuine amoeba crystals had disappeared and had been replaced by the characteristic recrystallized amoeba crystals. Centrifugation of the suspension with subsequent removal of the supernatant by pipetting and drying of the sediment yielded the preparation employed, about 70 ,~g [ll j. (c) ‘I’riuret was prepared as described by Schittenhelm and D’arnat il.3 by oxidation of uric acid with hydrogen peroxide in ammoniacal solution. It \ras recrystallized t\vice from lvater. ’ \Vc are very grateful

to Dr. B. \V. Grunhaum

for presenting

us with thrsr

crystals.

D. Carlstriim and K. Max Moller

OBSERVATIONS

Genuine amoeba crystals.-The morphology and the physical and optical properties of genuine amoeba crystals have already been described in detail [6, 7, lo]. Therefore, only some of the more important data need be given here. Usually the crystals form truncated tetragonal hipyramids having an average length of 2-5 p, a: b:c = 1 : 1: 4.3. No distinct cleavage. Density 1.74 [lo] or 1.640 & 0.005. No apparent birefringence. However, in 1.74-1.75 [16], n,= specially large, highly flattened bipyramids from Amoeba dubia it has now been possible to detect a faint birefringence when these plates are viewed edge-on, i.e., perpendicular to the c-axis. By comparison with small crystals of other materials having a known birefringence it can be concluded that the birefringence of the amoeba crystals does not exceed 0.003. Earlier X-ray diffraction data obtained from powder diagrams could not be interpreted [S, 7, 10, 141, and electron diffraction data clearly led to unit cell dimensions that were neither in agreement with the morphology of the crystals nor with the X-ray data. Therefore a new attempt was made to establish the crystallography of the genuine amoeba crystals. Powder diagrams were recorded with Ni-filtered Cu radiation in a 190 mm diameter precision powder camera. The use of thin specimens (diameter RS70 ,LA)and a line focus gave a very good resolution of the lines of capillary in the powder patterns. The CuKa,-a, doublet was thus resolved already at 0 = 20”. In order to eliminate errors due to film shrinkage and absorption, the amoeba crystals were mixed with a suitable amount of silicon powder (a = 5.43063 A), which served as an internal standard. Since it was not possible to record interplanar spacings longer than 7 A with this equipment, X-ray diffraction patterns were also recorded in a flat-film camera capable of resolving d-values of about 80 A. The specimen-to-film distance was 68 mm. From the diffraction patterns, which contained some 75 reflections, 50 reflections could be measured accurately. This was about twice the number previously reported [6,7, lo]. By applying the method described by De Wolff [18] it seemed as if a satisfactory indexing of the diagrams was obtained. The tetragonal unit cell dimension thus found were: n = b = 5.06 .& and c = 21.7 A. The agreement between observed and calculated sin2 e-was good; out of thirty possible reflections having sin2 0 below 0.1100, twenty-three were measured; none deviated by more than 0.0005 from the calculated value. In addition there were, in this range, two reflections which did not fit very well (deviations 0.0010 and 0.0013, respectively), and further three lines Experimentd

Cell Research 24

Amoeba crystals which could not be accounted for at all. These three “extra” reflections at 8 = 9.30”, 11.34” and 18.05” were all rather faint, and it was believed that they stemmed from impurities. Strongly birefringent particles, usually much smaller than the amoeba crystals themselves, were in fart observed in the purified amoeba crystal preparation. It was, however, doubtful if the small amount of this “impurity” really could account for the “extra” lines observed. All attempts to identify this supposed second phase from the cl-values of the “extra” lines were in vain. h strong support for the correctness of the unit cell dimension found came from the morphology of the crystals. The observed obtuse angle (153”) between the prismatic faces of the crystals \vas in perfect agreement with that calwlated (133”G’) for the planes (101) and (101). However, a diffraction pattern of a very small amount of amoeba crystals recorded in a micro-diffraction camera (specimen stationary and specimen-to-film distance 15 mm) revealed that all lines had the same degwrx of spottiness, i.c., even the “extra” lines were caused by particles of the same size as the amoeba crpstals. This finding at once made the indexing somewhat To obtain an indisputable check on the unit cell dimensions du t,ious. it \\-as therefore found necessary to record a single crystal diagram. ‘I’htb results of earlier attempts in this direction were not at all encouraging. and trustworthy electron diffraction data seemed unobtainable becauw 01’ thta tendency of the crystals to decompose quickly in the electron Iwarn. For this reason a special micro X-ray diffraction camera1 \vas designetl so that the specimen could be rotated in the micro-beam (,jO ~1 in diameter). Among a large number of amoeba crystals, one of the t)t~st-tic\,clopcti anti largtbst (length about 4 ,u) was enclosed in a thin Parlodion film. The film \vas cut in rectangular shape (0.5 x 1 mm) with the crystal in the centre SO that the long dimension (c-axis) of the crystal ran parallel as nearly as possible to the long edges of the film. The film was subsequently placrtl 011 a thin glass fiber running in the direction of the c-axis of the crystal. Rotation diagrams (t\vo revolutions per hour) were completely unsuccessful even when the exposure time exceeded three days. The reflections were not strong enough to compete \vith the background caused by scattered radiation. The pro~)lcm \vas solved by letting the crystal remain stationary in the beam for c~lrveu hours at a time . .After such an exposure the crystal was rotated about 2’) and a nc\v exposure was performed. Because of the slight divergence 01’ thtb hcam (about I”) a few (O-4) reflections \vere recorded each time. AllcI twenty-one exposures covering an angular range of about 45’, thirty-six ’ \Ve a~‘(’ much indebted (‘:LmPr;,.

to Dr. K.-A.

Omnell and I)r. J-I<.

Glas for the coustrurtion

of I his

D. Carlstriim and K. Max MBller reflections had been collected in all. When the enlarged micro-diffraction patterns were plotted on the same graph, the presence of four ro\v-lines 011 each side of the meridian were readily recognizable. Their positions were in perfect agreement with those predicted for a tetragonal unit cell having (x = b = 5.06 hi. The presence of one meridional reflection and the bending of the row-lines showed that the c-axis of the crystal had deviated about 13” from the axis of rotation. Because of this fact and because of the rather limited amount of reflections observed, it was not possible to identify any layer-lines properly. However, in three instances closely spaced pairs of reflections having the same hk-indices had been recorded. From the distance in the meridional direction between mutual reflections in such pairs it became evident that the length of the c-axis was of the order of 40-45 ,& instead of the presumed 21.7 A. A doubling of the c-axis dimension immediately brought the calculated sin2 19into perfect agreement with the observed sin2 0 derived from the powder patterns, and the “extra” lines could now be incorporated (Table I). The genuine amoeba crystals are thus tetragonal with the axes: n = b = 5.05 A, c = 43.40 A; a: b:c = 1 : 1 18.594. A sketch relating the morphological faces to the unit cell is seen in Fig. 1 A and IS. The absence of all (OOZ)-reflections, where 1 is not a multiple of 4, shows the presence of a fourfold screw axis. No other systematic absences were observed with certainty, and therefore the exact space group cannot be given. The volume of the unit cell is 1107 A3. Taking the density as 1.745 (average of earlier measurements) a molecular weight of 1164/n is obtained. In this case R has to be a multiple of four, and the molecular weight can thus be either 291, 146, 97 or 73. Recrystallized amoeba crystals and trillret.-X-ray data from powder as well as single crystals of recrystallized amoeba crystals were available several years ago, but since no substance fitting these data was found in the literature the nature of the compound was at that time obscure. Thanks to the work of Griffin [6, 7] it is now clear that recrystallized amoeba crystals and triuret (carbonyldiurea, C,H,N,O,, mol. wt. = 146.11) are identical. Triuret crystallized from aqueous solutions always occurs in the form of extremely thin plates with a perfectly squared outline. Crystals up to 0.5 mm across having a thickness of about 20 ,u have been obtained. In such “large” crystals it can be seen that the crystals have only one plane of symmetry, indicating that they are monoclinic. The poorly developed (110) faces of the edges are inclined 65-70” (calculated on the basis of the X-ray data: 68.2”) to the large (001) faces of the plates (Fig. 1 C). Sometimes (100) is also developed. There is a very pronounced and perfect cleavage along Experimental

Cell Research 24

3917

Amoeba crystals TABLE I. X-ray

Int.’

a Graded

diffrrrction

data from yenuine Sin2 Oobs A 10,000

amoeba crystals.

Sin2 Ocalc x 10,000

0‘ obs

d (ii)

4.0.5

10.91

50

50

x.17

5.42

202

202

X.X-1

5.01

236

233, 236

9.01

-1.92

245

2-16

VW

m&l) (00X) (100)

(101)

(loz

9.30

4.77

261

261

it031

9.6X

4.58

283

283

(104)

lO.li

4.36

312

312

\ 1O.i)

10.72

-1.14

346

3-16

(106J

11.34

3.92

387

3xi

it(E)

12.30

3.62

454

43-t

(nn.12)

12.G

357

466

165. 46X

1110) (111)

1’2 . i’,

3.49

487

38X

(109)

13.12

3.39

515

51.5

1111)

13.54

3.29

548

54x

(10.10)

13.92

3.20

579

578

(116)

1 1.1’

3.09

620

619

(lli)

14.96

2.984

666

667

(11X)

15.19

2.940

687

6X7

(10.12)

16.2‘2

2.758

780

7x0

(11.10)

(16. Ii)

(2.717)

(804)

X08

(00.16)

846,852

16.94

2.644

849

15.65

2.540

919

919

(17.79)

(2.521)

(933)

930, 933

17.89

2.507

944

943

(11.11)

110.14)

(Il.121 !200)

i201)

(202i

1X.0.5

2.486

960

9.58

(2031

18.25

2.460

981

9x0

(204)

(1X.53)

(2.424)

(1010)

1 orr9

18.81

2.389

1040

1041,1043

(10.16)

(206)

19.23

2.339

1085

10X&

(11.11)

(207)

19.77

2.277

1144

1084

1 l-43 11.63,

1166

(205)

(10.15) (2101 (211)

19.95

2.258

1164

XI.14

2.237

1186

1185

20.38

2.212

1213

1213

20.64

2.185

1243

1242,

124<5

(2151 120.10)

2n.93

2.156

1276

1273,

1276

(11.16)

22.12

2.046

1418

1418

(219)

22.62

2.003

1479

1478

(21.10)

23.14

1.960

1544

1544

(21.11)

124.301

(1.X72)

(1693)

lG95

(21.131

l-6

with

falling

intensity.

(209) (214) (216)

D. Carlstr6m and K. hfaz Moller

398 Table

I (cont.)

ht.’

6 6 5 5 6 6 6 6 6 6 6

O”obs

d (A)

Sin” O,,, x 10,000

Sin2 Bcalc x 10,000

(24.64) (24.82) 24.97 26.14 26.21 26.35 (27.10) (27.S7) 29.04 29.39 (29.82)

(1.848) (1.835) 1.825 1.748 1.744 1.735 (1.691) (1.648) 1.587 1.570 (1.549)

(1738) (1762) 1782 1941 1951 1970 (2075) (2185) 2356 2408 (2473)

1738 1758 1782 1939 1953 1971,1973 2073 2186,219O 2353 2408 2470,2474

(W (20.16) (10.22) (21.14) (225) (20.18) (21.16) (226) (21.17) (21.18) 120.20) (313) (30.10) (00.28) (30.11)

(100) which makes the crystal break easily into thin rods. Growth layers are seen on the (001) face. The acute angle between the cleavage plane and (001) was found to be 59-62” (calculated on the basis of x-ray data: 60.5”), but even in this case it was difficult to get accurate values because of the extreme thinness of the plates. Twinning was observed on (001). The density was measured by the pycnometer method with a petroleum fraction immersion medium: d = 1.738 g/ml (21°C). This is somewhat lower than the value 1.745 reported by Griffin [6, 71. Between crossed polarizers the triuret plates showed extinction parallel to the cleavage (100). Triuret is not uniaxial as stated earlier [6, 7, lo] but biaxial negative ( - ). The axial angle is, however, quite small, 2 V = 10 & lo, which may explain the fact that the conoscopic interference figure from plates lying flat on the (001) faces looks like an inclined uniaxial figure. The orientation of the indicatrix related to the morphological axes is such that % is parallel to 6, Y is parallel or very nearly so to the c-axis and X A c w 90”. Indices of refraction for sodium light (interference filter): a = 1.445 +_0.003, ,8 = 1.722 (calculated), y = 1.725 + 0.003. The birefringence is thus very high (0.280). The main indices of refraction directly observable when the plates are lying flat on the (001) face are tc’ = 1.533 +_0.003 (along the a-axis) and y. Single crystal X-ray patterns of recrystallized amoeba crystals as well as synthetic material gave an end-centered monoclinic unit cell having the dimensions: rr = 7.17 A, b = 7.14 A, c =21.69 a, ,5’=95.6”. Space group Cc or C 2/c. The relation between the morphological unit cell, c1= 7.17 A, Experimental

Cell Research 24

Amoeba crystals

390

h i.1-l A, c= 21.81 -4, /I =tiO .5” and the true unit cell is seen in Fig. 1 1). The xwlume of the unit cell is 1106 a3. This gives eight molecules of triuwt per unit cell. (Z = 7.96 for a density of 1.745 g/cm3.) DISCUSSION

‘I‘hc crystallographic investigations reported ahove sho\v that there is a very close and certainly not fortuitous relationship het\veen the unit cell tlim~nsions of the genuine amoeba crystals, on the one hand, and the I‘(~cryslallizrd ant’s, on the other. The volumes of the unit cells arc thus iticntic:ll.

Pig. t.-‘The morphology of a typical genuine amoeba crystal is seen in (A), where the indices within brackets refer to corresponding planes in the structure cell (B) having the dimensions: rc h = 5.05 A. c 43.46 A. A triuret crystal is shown in (C). and three alternative unit cells are pictured in (D). The true end-centered monoclinic cell having the axes, o -~7.17 ;\, h -7.1-l K. c 2J.69 PI, B mm 95.6”. is seen in the middle. Its relation to the morphological cell (to the right J is obvious. This cell has the same n- and b-axes as the true cell, but c -~ 24.81 .% and @ 60.3 From the end-centered true unit cell a smaller triclinic cell (to the left) can bc derived. It has the dimensions n = h 5.66 -4, c - 21.69 h, GC-B =94.0‘. y X9.7 ‘. Note the similarities between lhis cell and that of the genuine amoeba crystals.

Since the densities of these two compounds arc very nearly the same, anal assuming each cell to contain eight molecules, one arrives at the ronvlusion that they have identical molecular weights (mol. wt. = 146). The unit c*cll of triuret is end-centered monoclinic, and therefore it can be descrihvti equally well as a special case of a triclinic cell containing four formula units with n = h = n.O(i .i, c = 21.69 A, CI= fl = 91.0”, y = 89.7” (see Fig. 1 I)). A section cut through this cell perpendicular to the (.-axis gives a pcrfwt

400

D. Carlstrdm and K. Max Mailer

square 5.05 A, which is the same as the square formed by the (I- and b-axes in the genuine amoeba crystals. Since triuret is obtained by recrystallization of genuine amoeba crystals without the appearance of any other crystalline compound, it seems quite obvious that the substances are crystalline modihcations of the same. compound. It is true that it has not been shown that the cell content of the genuine amoeba crystals is not four, twelve or sixteen molecules instead of eight, but it is very hard to conceive of any compound of molecular weight 291, 97 or 73 which could yield triuret by dissolution in water. From the crystallographic point of view there seems to be every reason to assert that the genuine amoeba crystals are nothing but triuret. This concept has already been advanced by Griffin [6,7,8], who used a quite difIerent approach. The final proof of the nature of the amoeba crystals would naturally be the production in vitro of the genuine amoeba crystals. So far all attempts to transform triuret to a tetragonal form have been unsuccessful [S, 71. We have tried eight different procedures for the recrystallization: (1) H,O at fourteen different temperatures between O”C-100°C. (2) 99.9 per cent D,O (hydrogen-bonding properties differs from H,O; triuret solubility lower in D,O than in H,O), 100°C+230C, several weeks at 23°C. (3) 10 M LiBr in H,O (good solvent for denatured proteins) 65“~-+23’C, several weeks at 23°C. (4) 0.1 Msodium acetate buffer in H,O, pH 4.6, 1OO”C+23”C. (5) Saturated solution of NH,Br in H,O [I], cooled from 40°C to 23°C during two days and thereafter kept at 23°C for several weeks. (6) Dioxane (low dielectric constant) 65’C-t23’C, several weeks at 23°C. (5) Formamide (very high dielectric constant) 64’C+23’C, several weeks at 23°C. (8) High pressure, up to 9000 kg/cm2 at 22°C on dry crystals and crystals moistened with H,O. There are, however, several objections which can be raised against the above interpretation of the chemical nature of the genuine amoeba crystals. Some minor objections arise from physical as well as biological observations, but the most serious is the marked discrepancy between the elementary analysis of genuine amoeba crystals and the composition of triuret. From stereo-chemical considerations and the analogy with the known crystal structures of urea [171 and biuret [3, 5, 12, 131 it seems quite probable that the triuret molecule is almost planar. The high negative birefringence of synthetic triuret indicates a structure with planar molecules all stacked parallel to one another. Some preliminary calculations on such Experimental

Cell Research 24

401

Amoeba crsytals

a sheet structure seem to be in agreement with X-ray data. The reason for the very low birefringence of genuine amoeba crystals is not yet clear. Ho\l-ever, none of the differences in the physical properties (including IH spectra and temperatures of decomposition) really contradicts the vie\v that genuine amoeba crystals and crystals of synthetic triurrt are built up of the same kind of molecules. If the diflerences observed are caused only by a different arrangement of identical molecules or if the molecules themselves also are sterically different cannot be decided from the present data. The most serious argument against the idea that the amoeba crystals consist of triuret comes from the chemical analysis. In all five preparations have heen investigated carefully: A, B, C, 11, and I<. L4, 13, and C were prepared in onr \vay [l(i] and D and E in a slightly different manner [Y:. A, 13, and C had the same-density [lo, 161, A, IS, and E had the same IK spectrum [lo, 11, C, I), and I’ bad almost the same nitrogen content IlO, 141, and 1) and E had nearly the same carbon, hydrogen, and ash contents :lO, 11 . Each of the t\vo tlifferent modes of preparation thus gave reproducible results. ‘I%(~ identity of the tww types of preparations is indicated by the IK spectrum and the nitrogen analysis. Another way of testing the purity of the preparations \vas microscopic investigation. The amount of impurities wuld in this \\-a> impurities be estimated as approximately 5-10 per cent. So crystalline could be detected in the X-rap powder patterns of various preparations, which means that if any crystalline impurities \vere present the concentration of’ each phase could not exceed l-2 per cent. The ash of the genuine amoeba crystals, amounting to about 11 per cent of the total weight, has already been accepted as an impurity [lo]. This vie\\ is further confirmed by the present X-ray data. Even if the molecular \\cighl of the molecule composing the amoeba crystals were the highest possible. according to the crystallographic data namely 291, then the molecular lveight of the ash could not exceed 32 if the ash were part of the molecule. This is an unacceptable \-alue, especially considering its content of phosphortIs (10.8 per cent) ill]. Disregarding the ash, one might also attribute the difference in elementary composition between genuine amoeba crystals (C z 31.3 per cent, H = 4.89 per cent, S = 29.9 per cent) and triuret (C ~=2A.i per cent, H = 1.14 per cent, N = 38.4 per cent) to impurities. ‘These must then have a very low nitrogen content, since an assumption of contamination with a substance of similar nitrogen content as protein would require an amount of impurities far exceeding that observed by microscopy. Haying no\v considered some of the physical and chemical objections to the crystallographic interpretations a few biological ones \\-ill bc added. Esperimrntol

Ceil

Resrnrch

24

402

D. Carlstriim and K. Max Mdler

The following biological observations are considered relevant. If amoebae (Chaos chaos or Amoeba proteus) are crushed in the presence of a comparatively small volume of water (ten to one hundred times the volume of the amoebae), all genuine amoeba crystals disappear, and the strongly birefringent triuret crystals appear in the course of fifteen to thirty minutes at around 23°C. Afterwards, these crystals may disappear slowly (if the water volume is too large). In contrast, isolated genuine amoeba crystals may be stored in glass-distilled water (pH about 6) or in aqueous acetate or citrate buffer (pH 4.7) at 23’C for about four months without visible loss of crystals apart from that occurring until the solution is saturated. No triuret crystals are formed. However, the smaller crystals slowly disappear, while the larger ones increase in size. In this way the size distribution grows more uniform, but still the crystals are small. How far such an experiment can be extended depends exclusively on the occurrence of bacterial infection. A considerable number of morphologically ditferent bacteria degrade genuine amoeba crystals as well as triuret; in this way the crystals simply disappear. The above observations indicate that amoeba cytoplasm is able to catalyze the conversion of genuine amoeba crystals to triuret crystals. This, in conjunction with the fact that nothing but triuret is formed by recrystallization of isolated genuine crystals from water of about 50°C [ll], of 60-70°C [5, 61, or of 100°C [ 10, 1 I], suggests that the triuret is formed by a hydrolysis of the substance of the genuine crystals promoted by the catalytic action of the amoeba cytoplasm or by the elevated temperature. Amoeba dubia is an organism that possesses both types of crystals, viz., in the first place, typical genuine amoeba crystals, bipyramids which look like plates and appear isotropic when viewed flat, and in the second place, typical plate-shaped triuret crystals with a strong birefringence [4, 71. In this organism the relative frequencies of “isotropic” and birefringent crystals depend on the nutrition [4]; feeding with tetrahymena leads to preponderant formation of birefringent crystals, while feeding with a mixture of organisms (flagellates, paramaecia, and numerous other protozoa) yields mainly “isotropic” crystals. If A. dubirr is grown in a medium saturated with synthetic triuret, it contains chiefly exceptionally large birefringent crystals [4]. In view of the S-ray crystallographic data, none of the above two physical and two biological observations really challenge the hypothesis of identity of the molecules entering into the two crystal forms when the criteria of purity of the native crystals are taken into consideration. The differences in IR spectra and in temperatures of decomposition may be due to the difference in sterical configuration. IR spectra of triuret are identical with those found Experimental

Cell Research 24

Amoeba crystals

103

\\ith the residue obtained when genuine amoeba crystals are dissolved in hot water and the water is allotted to evaporate. This fact vannot he PSplaincd on the assumption of non-identity of the molecular building-blocks of the crystals, unless it is postulated that the hydrolysis by-product formed is volatile or has an extremely lo\v infrared absorption over the entire rangt’ studied. On the other hand, it must be admitted that the accompanying substances that are responsible for the difference in elementary composition must also bc volatile or hare a wry low infrared absorption or an absorptiotl spwtrum not very diRerent from that of triurct. The morphological change catalyzed by the amoeba cytoplasm might vt’r> xvcll be a sterical rearrangement. If genuine amoeba crystals are placed in a saturated triuret solution containing triuret crystals, they disappear slo\vly. Attempts to establish, by qualitative experiments, Avhether this phenomenon is due to the solubility of the crystals in a medium that is not saturated with Iheir substance or to a rhange of a thermodynamically less stable vrys~al structure to a more stable structure have not been suc~~cssf’ul. “kotropic”, gcnuinr ‘i’hc situation encountered in A. drrhirr containing amoeba crystals and strongly birefringent triuret crystals in the same r\-IOplasm tlocs not speak against the hypothesis of identity because the cytoplasm is not an equilibrium system, but a steady state system. It has furthermow Iwctn definitely established that in certain fishes and rcptilcs statcwonia consisting of calcium carbonate occur simultaneously in lhe same biologic*al ctnvironmrnt as calcite and vateritc or as calcite and aragonite ‘2:. I-io\\-c~vc~r, no obvious explanation of the dependence on thr nutrition van tw givvll.

SUMMARY A reinvestigation of the crystals found in the cytoplasm of Ar~oehrr /wo/ctrs has revealed that they probably consist of a tetragonal form of triurct (carbongtdiurea). This conclusion is mainly based on data obtained from S-la! diffraction and polarized light microscopy. The more stable monoclinic form of triuret obtained by synthesis or by recrystallization of gcnuints amoeba crystals from water has also been studied. Some of the dilferenccs observed between genuine amoeba crystals and ordinary pure triuwt cannot be csplained by a difl’erent sterical arrangement of the molecules. The! arc therefore considered to be caused by impurities. N’hether these impurities are introduced during the course of isolation (non-aqueous gradient) of the amoeba crystals or whether thev are present in r~irw is not kncnvn.

404

D. Carlstr~m and K. Max Mdler

The authors wish to express their gratitude to Professor H. Holter and Dr. C. Chapman-Andresen and Dr. H. Ringertz for their interest, advice and assistance during the course of this investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18.

567 (1933). BIJNN, C. W., Proc. Roy. Sot. LondonA141, CARLSTRBM, D., Unpublished results. CAVALCA, L., NARDELLI, M. and TAVA, G., Acta Cryst. 13, 594 (1960). CHAPMAN-ANDRESEN, C. and MAX MILLER, K., Unpublished results. FREEMAN, H. C., SMITH, J. E. W. and TAYLOR, J. C., Nature 184, 707 (1959). GRIFFIN, J. L., Ph.D. Thesis, Princeton University 1959. University Microfilms, Inc., Ann Arbor, Michigan, No. 59-5183. __ J. Biophys. Biochem. Cytol. 7, 227 (1960). __ Biochim. et Biophys. Acta 47, 433 (1961). GRUNBAUM, B. W., Compt. rend. trav. lab. Carlsberg 32, 179 (1961). GRUNBAUM, B. W., MAX MBLLER, K. and THOMAS, R. S., Expfl. Cell Research 18, 385 (1959). I_ Compt. rend. trav. lab. Carlsberg. To be published. HUGHES, E. W., YAKEL, H. L. and FREEMAN, H. C.. Acta Crust. 14, 345 (1961). ’ KUMLER, W. D. and LEE, C. M., J. Am. Chem. Sot. 82, 6305 (1960): MAX MILLER, K. and THOMAS, R. S., Compf. rend. trav. lab. Carlsbero. To be nubhshed. 1 SCHITTENHEL~, A. and WARNAT, K.; Z. physiol. Chem. 171, 174 (1927). THOMAS, R. S., Compt. rend. trav. lab. Carlsberg 32, 155 (1961). VAUGHAN, P. and DONOHUE, %J., Acta Cryst. 5, 530 (1952). DE WOLIV, P. M., Acta Cryst. 10, 590 (1957).

Experimental

Cell Research 24