Microfilaments from amoeba proteins

Experimental Cell Research 65 (1971) 7-16

M I C R O F I L A M E N T S F R O M A M O E B A P R O T E I N S

JOAN MORGAN

Department of Biology as Applied to Medicine, The Middlesex Hospital Medical School, London W1, UK

SUMMARY

Bundles of microfilaments have been obtained from a fraction of homogenised amoebae cells after the addition of EDTA. These bundles are made up of many single microfilaments 30 40 A in diameter. An attempt has been made to obtain the protein subunit of these microfilaments. In the detergent Sarkosyl, a subunit of sedimentation constant 2.0 • 10 -I8 and mol. wt approx. 42 500 has been found. The amino acid composition of this protein is similar to that of muscle actin, plasmodium actin and microtubule subunit protein. On removing the Sarkosyl, the protein reaggregates into bundles of microfilaments like those originally formed with EDTA.

In the course of our inevstigation on the structural basis of amoeboid movement, an at tempt has been made to isolate and identify the structures responsible for mobility. The starting point for this work was the isolation of a crude cytoplasmic fraction which on the addition of adenosine triphosphate (ATP) 1 showed streaming movements and contraction. In addition the contracted regions re- vealed the presence of filaments [35, 40, 41]. When EDTA was added to a more purified fraction, bundles of microfilaments appeared [22]. Because the preparation with EDTA is much easier and more reliable to obtain than that with ATP, the work here has been con- cerned only with those filaments formed with EDTA, though it is hoped that this information can be related to the filaments formed with ATP as well.

Abbreviations used: ATP, adenosine triphosphate; EDTA, ethylene diamine tetracetic acid; PTA, phos- photungstic acid; PCA, perchloric acid; PCMB, p-chloromercuri benzoate; SDS, sodium duodecyl sulphate.

The microfilaments formed on addition of EDTA have been isolated and an at tempt made to obtain and examine their protein subunit. However, the strong tendency of the microfilament subunit protein to aggregate and the small amount of material has made these studies difficult.

MATERIALS A N D M E T H O D S

Bulk isolation of microfilaments

A homogenate of amoebae cells was prepared as described in Thompson & Wolpert [35]. Membrane free homogenate was centrifuged for 2 h at 150 000 g and this supernatant was used for obtaining the microfilaments. EDTA was added to the supernatant at a final concentration of 3 mM and left at room temperature for 15 rain. Microfilaments appeared as a white flocculent precipitate, which was sedimented by centrifugation at 35 000 g for 1 h. The microfilaments were extracted from the pellet by gently homogenis- ing it with an appropriate volume of 1 M KC1. After 5 h at 4~ the undissolved material was removed by spinning at 35 000 g for 30 rain. The formation of microfilaments could be followed under the microscope by adding EDTA to a drop of supernatant on a slide [22].

Exptl Cell Res 65

8 Joan Morgan

Examination of microfilaments in the electron microscope The isolated microfilaments were examined using negative staining methods. Samples were prepared on carbon/Formvar-coated grids and negatively stained with 2 % PTA according to the technique of Huxley & Zubay [15].

Samples of pelleted material were fixed for 1 h in 2.5 % glutaraldehyde in cacodylate buffer pH 7.0, containing 7 mM calcium, post-fixed in 1% osmium tetroxide in the same calcium-containing buffer and then processed in the usual way.

The grids were examined in an AEI EM6 and Philips 300E microscope. Measurements of the microfilament sizes were made on the negative with the aid of a dissecting microscope.

Examination of the extract of microfilaments (a) Protein was estimated by the Lowry method [18] using Wellcome Chemical Control serum as a standard (Burroughs Wellcome & Co., Ltd.).

(b) Deoxyribonucleic acid and ribonucleic acid were estimated using the standard techniques described in Monro & Fleck [21].

(e) Carbohydrate, as glucose, was measured by the Anthrone method following the procedure described by Trevelyan & Harrison [5].

(d) Phospholipid was measured by Allen's method [1].

(e) Bound nucleotide was estimated using the method described by Newton & Rothschild [23]. An equal volume of cold 0.5 % PCA was added to the sample at 0~ The precipitate was removed and the supernatant neutralised with 1 N KOH. The precipitate of perchlorate was sedimented and the nucleotides estimated in the supernatant from their ultraviolet absorption spectrum (see P'Abst. Labora- tories Circular OR-10, "Ultraviolet absorption spectra of 5"-nucleotides" PL Biochemicals, Milwaukee, 1961).

In addition, a sample was applied to a paper chro- matogram. Guanine and adenine nucleotides were used as standards. An ascending solvent system was used consisting of 100 vol isobutyric, 55.8 vol water, 4.2 vol ammonia and 1.6 vol 0.1 M EDTA. The chromatograms were examined under UV light for the presence of nucleotides.

( f ) Free sulphydryl groups were measured by the PCMB titration method of Boyer [8] as described in Benesch & Benesch [5].

Attempts to obtain the subunit of the microfilament protein The extract of microfilaments was dialysed against various solvents in an attempt to minimise aggregation and obtain the subunit. In each case the mierofilament protein in 1 M KC1 was dialysed for 24 h with several changes in the cold against the particular solvent. The effects were followed by ex- amining the sedimentation pattern in the analytical ultracentrifuge.

Examination of the protein subunit of the micro filaments (a) Examination in the ultracentrifuge: Sedimentation velocity experiments were carried out in a Spinco Mo- del E analytical ultracentrifuge equipped with Schlie- ren optics and accurate temperature and speed controls. The plates were measured with the aid of an enlarger coupled to a PCD digital data recorder. Sedimentation constants were calculated and cor- rected to standard conditions as described in Schachman [30, 31].

The molecular weight was determined by the Archibald method as described in Schachman [30, 31]. The determination was made in 0.5 % Sarkosyl in 0.01 M Tris HC1, pH 8.5, containing 0.01% mercaptoethanol, and using a Spinco Model E analytical ultracentrifuge, with Schlieren optics, the plates were measured as described above. The calcula- tions were done on the London University Atlas Computer using a programme written by Mr M. R. B. Clarke (Institute of Computer Science, London University) to directly process the tapes produced by the enlarging apparatus.

Molecular weight was calculated from the slope of a plot of

l (dc) 1 f 2(de)~ x dxx against r -(dx)ax,

where x is the distance from the centre of rotation and dc/dx the concentration gradient at the meniscus and cell bottom respectively. For a completely homogeneous solution, two straight lines of equal slope but opposite sign will be obtained from the meniscus and cell bottom. The slope is equal to - M [ ( 1 - Vg)/RT]o9~, where M is molecular weight, P partial specific volume, ~ the density, ~o speed of rotation, R the gas constant, and T the absolute temperature. For heterogeneous material, the heavier particles go to the bottom of the cell and the apparent molecular weight increases, giving a curve of numerically increasing slope. In practice the values of (dc)/dx at the meniscus and cell bot tom were determined by extrapolation. An estimate of Co is not necessary for calculating the molecular weight by this method. The partial specific volume was calculated from the amino acid analysis as described by Cohn & Edsall [10].

(b) Examination of the subunit using polyacrylamide gels: It is possible to estimate the molecular weight of a protein in SDS using polyacrylamide gels containing this detergent [27, 32]. The protein-SDS complexes are negatively charged and move towards the anode and mobility is not related to the charge on the native protein. There is a linear relationship between moelcular weight and mobility, mobility being inversely related to molecular weight [27, 32], Con- sequently, with a suitable choice of protein markers it is possible to estimate the molecular weight of a protein from its mobility. Here the detergent Sar- kosyl (sodium lauryl sarcoseine) was used instead of SDS and it was found that a plot of log molecular weight against mobility gave a linear relationship for the proteins and gel concentration used.

Exptl Cell Res 65

Microfilaments from amoeba proteins 9

Preparation of gels: Sarkosyl was included in the gels and running buffers. The gels were 7 % acrylamide in 0.1 M phosphate buffer pH 7.0 containing 0.5 % Sarkosyl. The amount of BIS used was 2.6 % that of acrylamide. The final concentration of persulphate was 0.07% and TEMED 0.06%. Samples were allowed to polymerize in the dark. Electrophoresis was carried out in an apparatus like that of Davis [11]. The sample containing sucrose was layered directly on top of the gel. Bromophenol blue was included as a marker in each gel tube. The gels were approx, 7.5 cm long and they were electrophoresed for 3�89 h at 8 volts/cm. The running buffer was 0.1% Sarkosyl in 0.01 M phosphate pH 7.0. Gels were removed by running under water, stained for l h in 1% Amido Schwartz in ethanol (5 vol) glacial acetic acid (1 vol) and water (7 vol) and destained in ethanol, acetic acid, water (5 : 1 : 7).

Measurements of band positions were made using a Joyce Loebel Chromoscan. The position of bromophenol blue and the gel length were noted before staining and a correction made for the change in length occurring during staining and destaining. The migration of bromophenol blue was used as a reference within each gel tube giving a mobility for the protein relative to bromophenol blue. The mobility of cytochrome c was used as a reference for each experiment. Thus, the protein mobilities were finally expressed relative to cytochrome c. Relative protein mobility = Rf bromophenol blue for protein/ Rj bromophenol blue for cytochrome e.

Preparation of samples: Microfilament protein was dialysed against 0.5% Sarkosyl 0.01% mercaptoethanol in 0.01 M phosphate pH 7.0 and the marker proteins were dissolved in the same solvent. Crystal- line proteins with a range of molecular weights from 12400 67000 were used and the values of their molecular weights were taken from Andrews [2], Apella & Market [3], Ingram [16], Maehly [19] Tanford & Lovrien [341.

Each sample for electrophoresis consisted of 25 #1 protein (approx. 60/~g) 25/~1 of 40 % sucrose and 25/~1 of bromophenol blue.

(c) Amino acid analysis of the subunit: The sample of microfilaments in Sarkosyl was dialysed against water and freeze-dried. There was insufficient material for nitrogen or ash determinations to be made on the sample and the results are expressed as moles of amino acid per 105 g recovered amino acid, and the number of residues per 105 g of protein calculated from them. The sample was hydrolysed for 24 h at 110~ in a sealed tube under vacuum and the analysis carried out on a Beckman Model 120C autoanalyser. (The estimation was carried out by Dr S. M. Part- ridge, Meat Research Institute, Langford, Bristol.)

RESULTS

Our previous work had shown that the pellet of microfilaments formed with EDTA contained vesicular, membranous and lipid

material in addition to the bundles of microfilaments [22]. Extraction of this pellet with 1 M KC1 resulted in the removal of the filamentous structures and only lipid and membranous material remained in sections of the insoluble pellet (fig. 1 a, b). Examination of the protein content of the soluble supernatant showed that 1 M KC1 extracted 65-78 %, 0.6 M KC1 extracted 40-50 %, and 0.1 M KC1 only 36 %. The yield of protein obtained in this way was very low. From 16 million cells usually 1.0-1.5 mg of 1 M KCI extracted protein were obtained. This protein represented 1.0-1.5% of the total protein present in the membrane-free homogenate. This appears to be a very small amount of cellular protein associated with motility but Hoffman-Berling [14] found that the contractile protein from sarcoma cells represented only 0.1% of the dry weight of the cell, and Wolpert [40] calculated that prob- ably relatively few filaments would be needed to generate motile forces.

Examination of the 1 M KC1 extract in the electron microscope by negative staining showed a few bundles of microfilaments as described previously [22] as well as what appeared to be microfilaments 60-70 A in diameter (fig. 2). The impression was gained that such microfilaments were helically coiled. Satisfactory negatively stained preparations were hard to obtain, because of the difficul- ties encountered when trying to stain in the presence of salt. When the salt concentration was lowered the microfilaments re-aggregated into bundles similar to those originally formed with EDTA.

The 1 M KC1 extract contained no detect- able DNA, 3.0 % RNA, 1.4 % carbohydrate as glucose, 0.67% phospholipid, and 13.5 free-SH groups per 100 000 g of protein. All these values are expressed as a percentage of total protein present. Bound adenosine and guanosine nucleotides were sought in the

Exptl Cell Res 65

10 Joan Morgan

Fig. 1. (a) Electron micrograph of a section through the EDTA-induced gel, showing fibrous bundles and membranous contaminants. The fine network of microfilaments can also be seen. x 25 000. (b) Electron micrograph of a section through the insoluble remainder of the EDTA gel after extraction with 1 M KCI.

Exptl Cell Res 65

Mierofilaments from amoeba proteins 11

Fig. 2. Electron micrograph of a single microfilament found in the l M KC1 extract of the EDTA-induced gel. x 120 000.

preparation of microfilaments in 1 M KC1. None could be detected even in very fresh samples. Due to the small amount of material available and the strong tendency of the protein to aggregate, no further purification steps were taken and the preparation of microfilaments in 1 M KC1 formed the starting point for further investigations.

In the analytical ultracentrifuge the preparation in 1 M KC1 gave a slow-sedimenting peak which rapidly became highly asymmetric and elongated, suggesting the presence of subunits which were aggregating into larger structures. This would agree with the electronmicroscope evidence, where some filamentous structures could be found.

A variety of solvents were tried in order to obtain a homogeneous preparation of the subunit. In 2 M lithium chloride, 1 M potas- sium thiocyanate, 4 M urea aggregation still continued. Maleation with maleic anhydride [9] did not minimise aggregation. This suggested the importance of disulphide bonding. Following Stephens' [33] experience with microtubule protein, Mersalyl, Sarkosyl and PCMB were tried. In both 50 mM Mersalyl and 0.5 % Sarkosyl in 0.01 M Tris/HC1 pH 8.5 containing 0.01% mercaptoethanol, aggregation was minimised and reasonably sym- metric peaks obtained (fig. 3 a, b). The estimation of the sedimentation constant in Mersalyl was made at 18~ and a S~. 20 of

Fig. 3. (a) A trace of the sedimentation pattern of microfilament protein in 50 mM Mersalyl, protein concentration 25 mg/ml. Schlerien bar angle, 50 ~ Photograph taken 90 rain after reaching speed. (b) A trace of the sedimentation pattern of microfilament protein in 0.5 % Sarkosyl in 0.01 M Tris-HC1 pH 8.5 containing 0.01% mercaptoethanol. Protein concentration, 4.6 mg/ml, Schlerien bar angle, 70 ~ Photograph 45 min after reaching speed.

Exptl Cell Res 65

12 Joan Morgan

Fig. 4 (a, b). Electron micrographs of re-aggregated bundles of microfilaments formed after dialysing away the Sarkosyl. Magnification (a) x 160 000; (b) x 180 000.

1.8 • l0 -la was obtained. Lowering the concentration of Mersalyl to 25 mM produced a highly asymmetric peak. In 0.5% Sarkosyl a S~ ~0 of 2.0• -13 was obtained, the measurement being made at 18~ Due to the small amount of material available it was not possible to carry out the number of investigations necessary for obtaining these sedimentation constants at infinite dilute. When salt 0.1 M KC1 was included in the Sarkosyl solution, in order to minimise electrostatic effects the peak became asymmetric and the salt seemed to be promoting aggregation.

When Sarkosyl was removed by dialysis

Exptl Cell Res 65

against distilled water, large bundles of 30-40 A microfilaments were formed. The individual microfilaments appeared beaded and the bundles seemed identical with those originally formed with EDTA (fig. 4a, b).

An estimate for the molecular weight of the subunit in Sarkosyl was made using the analytical ultracentrifuge and polyacrylamide gel electrophoresis. From the analytical ultracentrifuge investigation, readings obtained from the meniscus gave a straight line, which was used for calculating the molecular weight. The data from the cell bottom gave a very curved line indicating that aggregates were still present. Using a value of 0.733 for

~', calculated from the amino acid analysis a mol. wt of 42 600 was obtained for the subunit.

From the electrophoresis experiments a plot of relative mobilities of certain proteins against the log of their molecular weights was obtained (fig. 5). Each data point represents an average of at least four electrophoresis experiments and the line shown was fitted to the points by the method of least squares. The microfilament protein subunit gave a major slow-moving band a minor faster- moving band which could sometimes be seen to split into two (fig. 6). The mol. wt calculated for the major band was 42 000 and the minor ones 21 000 and 17 000 respectively. These minor bands may arise from some contaminating proteins, or re- present degradation of the major band.

Since in the analytical ultracentrifuge only a single peak was obtained, it is perhaps reasonable to conclude that they are degra-

1,0 Cytc ~ g RNAase .o Myg. ty

0.6 Chy. ~ " " ~

Perox. ~ ~ B Perox. ~

0.2 SA

Cat.~

4'.0 414 ' 4'.8

Fig. 5. Abscissa: log mol. wt; ordinate: relative mobilities.

Plot of relative mobilities of proteins against log molecular weight.

Protein Molecular weight

Cytc, Cytochrome c from horse heart 12 400 RNAase, Ribonuclease from ox pancreas 13 700 Myg, Myoglobin from sperm whale 17 800 Hb, Haemoglobin monomer (bovine) 17 000 SBT, Soya bean Trypsin inhibitor 21 500 Chy, Chymotrypsinogen 25 000 LDH, Lactic dehydrogenase rabbit muscle 32 000 Perox, Peroxidase 40 000 BSA, Bovine Serum Albumin 67 000 Cat, Catalase 32 000


*Gel end

Major peak

0 r

CQ 5"

Fig. 6. Trace of stained polyacrylamide gel after electrophoresis of microfilament protein in Sarkosyl. The major peak corresponds to the mol. wt value of 42 200.

dation products or at least only a minor contaminant, which might be binding stain strongly.

The results of the amino acid analysis are shown in fig. 7. Microfilament protein has a high content of dicarboxylic acids, especially glutamic acid, which makes up over 25 % of the total amino present. It is also high in

Table 1. Amino acid analysis of microfilament protein

Residues per 105 g Amino acid of protein

Lysine 72.4 Histidine 23.9 Ammonia Arginine 64.2 Aspartic acid 101 Threonine 60.7 Serine 57 Glutamic acid 152 Proline 41.27 Glycine 45.5 Alanine 56.7 Cysteine 13.5 Valine 55.5 Methionine 12.9 Isoleucine 63.2 Leucine 83.2 Tyrosine 49.6 Phenylalanine 48.5

Exptl Cell Res 65

14 Joan Morgan

arginine and lysine and in the sulphur- containing amino acid, cysteine.

DISCUSSION

The extract of microfilaments in 1 M KC1 showed strong tendencies to aggregate and it was difficult to find a solvent in which aggregation was minimised and the subunit could be obtained. In Sarkosyl a subunit was obtained, which had a sedimentation constant of 2.0 • 10 -1~, a molecular weight of 42 500 approximately and a partial specific volume of 0.733. The amino acid analysis of the microfilament protein shows strong resem- blances to the data obtained for muscle actin, plasmodium actin, microtubule subunit protein, and neurofilament protein (see Stephens [33]) for table of amino acid compo- sitions). No bound nucleotide could be detected which is in contrast to other proteins associated with motility, e.g. actin, plasmodium actin, or microtubule subunit protein, which have bound adenine and guanine nucleotides. However, bound nucleotide, if present, could have been removed by EDTA in the original formation of the microfilaments, since with G-actin EDTA removes nucleotide [4].

It was interesting to find that on removing the Sarkosyl, microfilaments 30-40 A in diameter similar to those originally formed with EDTA appeared and it was possible to correlate the information on the size of the protein subunit with this electronmicroscope information. From the molecular weight estimates and the sedimentation data a hydr- ated spherical protein subunit would have a calculated radius of between 40-50 A and so it does not seem unreasonable tha the protein subunits, by aligning end-to-end, could form the 30-40 A microfilaments seen in the electronmicrographs.

As reported in our earlier work various

Exptl Cell Res 65

filamentous structures have been found in extracts of amoeba cytoplasm [22, 40, 41], and other workers have also found filaments in sections of amoebae and other motile cells. In Amoeba proteus it seems possible that the 30-40 A microfilament examined here is the basic microfilament and would correspond to the network of finer filaments seen in sections of the ATP- and EDTA-induced gel [22, 41], and by lateral aggregation they could give the larger filaments of the EDTA gel [22]. In the ATP-induced gel, shorter 120--150 A filaments are present in addition to the 30-40 A microfilaments [41]. These may be formed from the small microfilaments or they may be another type of filament. There is a certain amount of evidence to suggest that all the microfilaments arise from the same precursor. In the formation of the two gels, neither occurs in the cold, and both need warming to room temperature for the appearance of filamentous structures. Also colchicine (3 raM) inhibited neither reaction. It was found if the ATP reaction was carried out on a sample of membrane-free homoge- hate and this sample was then centrifuged for 1 h at 150 000 g to sediment the cell compo- nents and the ATP-induced filaments, the EDTA reaction could not be obtained on this supernatant.

Filamentous structures similar to the microfilaments found by us have been found in sections of various amoebae cells and in other cells showing amoeboid movement and cytoplasmic streaming, so giving a certain amount of morphological evidence for asso- ciating these structures with the generation of motive force. Filaments 120-150 A long, like those of the ATP-induced gel have been found in Chaos chaos [24] in Amoeba proteus [28, 38] and in Trichamoeba [6]. It is interesting to note that the presence of these 120-150 A filaments may be related to the fact that contraction has occurred [35, 41].

This would to some extent be supported by the presence of similar filaments in situations in which mechanical injury may have caused cytoplasmic contractions, e.g. the micro- injection of fixative and the torn plasma- lemma [24, 38]. It has been suggested that similar filaments in Trichamoeba are con- nected with contractions at the rear of the cell [6] and, furthermore, that the 120-150 filaments are a stable component [28].

Also, microfilaments, like those isolated here and bundles of these microfilaments, have been found in a variety of cells, all of which are involved in motility of some type. Bundles of these microfilaments have been found in cells of Chaos chaos [24], in the pseudopods of Difflugia, where it has been suggested they may be contractile [39]. They have been found in association with cytoplasmic streaming in Physarum [29, 37], Nitella [25] and in oat coleoptile cells [26]. In addition to the morphological evidence link- ing these filamentous structures with motility, there is also the fact that in Physarum, F-actin has been isolated which is very similar in appearance to the microfilaments found in sections of the plasmodium [13]. Consequently, it is interesting to see that the isolated microfilaments from amoebae are very similar in appearance to those in sections of Physarum and, further, the microfilament protein subunit and plasmodium actin are similar in size and amino acid composition.

In view of the involvement of actin and plasmodium actin in movement and the possibility that the amoeba microfilaments may also be involved in generating motive forces it is interesting that there are resem- blances between the actins and microfilament subunit protein. The molecular weight of microfilament subunit protein is a little lower than that of plasmodium actin, but close to a recent estimate for actin of 47 600


[15]. The amino acid composition of the actins and microfilament protein shows the same pattern of high levels of the basic amino acids, dicarboxylic acids, especially glutamic acid and of the sulphur-containing amino acids. Actin does not bind colchicine [7] and colchicine does not inhibit filament formation with ATP or EDTA. There are differences: the method of preparation with EDTA is quite unlike that of both actin proteins. However, in isolated Plasmodium droplets, Wohlfarth-Botterman [37] has reported that injection of EDTA causes an increase in the number of microfilaments, and this may be a similar situation to the formation of the EDTA gel here. There is also a lack of bound nucleotide in the amoeba microfilaments whilst actin has bound ATP. This lack of nucleotide may be due to its removal during microfilaments formation, which could be due to EDTA or the nucleotide may be labile and easily lost. In the case of microtubule subunit protein the GTP is labile [33] and Davison [12] suggests that nucleotide may be lost during the isolation of neurofilaments. Binding experiments with labelled nucleotides would obviously be very useful in solving this problem. Lastly, though microfilament protein readily aggregates it does not form the two-stranded structure of F-actin.

Certain similarities have emerged between microfilament protein and neurofilaments. The bundles of microfilaments formed with EDTA and those reaggregated from Sarkosyl are very similar to the isolated bundles of neurofilaments from the leech nervous system [20]. Also, in molecular weight, amino acid composition, absence of nucleotide and the strong tendency to reaggregate, microfilament protein is similar to neurofilament protein from squid axon [12].

I should like to thank Professor L. Wolpert for his advice and encouragement throughout this work and

Exptl Cell Res 65

16 Joan Morgan

the Medical Research Council for financial support. I should also like to thank Mrs Betty Pearson and Mrs Christine Howatson for culturing the amoebae, Dr G. Mills of The Courtauld Institute of Bioche- mistry, The Middlesex Hospital Medical School and Dr E. G. Richards of the Department of Biophysics, King's College, for help with the analytical ultracentrifuge experiments and Dr B. Richards of G. D. Searle & Co. Ltd., for use of their electron microscope.

R E F E R E N C E S

l. Allen, R J L, Biochem j 34 (1940) 858. 2. Andrews, P, Biochem j 91 (1964) 222. 3. Apella, E & Market, C L, Biochem biophys res

commun 6 (1964) 171. 4. Barany, M, Finkelman, F & Therallit, A T, Arch

biochem biophys 98 (1962) 43. 5. Benesch, R & Benesch, R E, Methods of biochem

analysis (ed G Glick) 10 (1962) 43. 6. Bhowmick, D K, Exptl cell res 45 (1967) 570. 7. Borisy, G G & Taylor, E W, J cell biol 34 (1967)

525. 8. Boyer, P D, J Am chem soc 76 (1954) 4331. 9. Butler, P J G, Harrison, L J, Hartley, B S &

Leberman, R, Biochem j 103 (1967) 78 p. I0. Cohn, E J & Edsall, J T (eds). Proteins, amino

acids and peptides. Reinhold, New York (1943). 11. Davis, B J, Ann NY acad sci 121 (1964) 404. 12. Davison, P F, Neurosci res prog bull 6 (1968)

176. 13. Hatano, S, Totsuka. T & Oosawa, F, Biochim

biophys acta 140 (1967) 109. 14. Hoffman-Berling, H, Comparative biochemistry,

(ed M Florkin & H S Mason) vol. 2, p. 341. Academic Press, New York (1960).

15. Huxley, H E & Zubay, G, J mol biol 2 (1960) 10. 16. Ingram, V M, Nature 183 (1959) 1795. 17. Johnson, P, Harris, C L & Perry, S V, Biochem j

(1967) 361. 18. Lowry, O H, Rosebrough, N T, Farr, A L &

Randall, R J, J biol chem 193 (1951) 265. 19. Maehly, A C, Methods in enzymology (ed S P

Colowick & N V Kaplan) vol. 2, p. 80. Academic Press, New York (1955).

20. Metzuals, J, J cell biol 34 (1967) 690. 21. Monro, H N, Fleck, A, Methods of biochemical

analysis 14 (1966) 113. 22. Morgan, J, Fyfe, D & Wolpert, L, Exptl cell res

48 (1967) 194. 23. Newton, A A, Rothschild, Lord, Proc roy soc,

London, Ser B 155 (1961) 183. 24. Nachmias, V T, J cell biol 23 (1964) 183. 25. Nagai, R & Rebhun, L I, Ultrastruct res 14 (1966)

571. 26. O'Brien, T P & Thurmann, K V, Proc natl acad

sci US 56 (1966) 888. 27. Peel, D, Biochem j 108 (1968) 51 p. 28. Pollard, T D & Ito, S, Personal communication. 29. Rhea, R P, J ultrastruct res 15 (1966) 349. 30. Schachman, H K, Methods in enzymology (ed

S P Colowick & N O Kaplan) vol. 4, p. 32. Academic Press, New York (1957).

31. - - Ultracentrifugation in biochemistry. Academic Press, New York (1959).

32. Shapiro, A L, Vinuela, E & Maizel, J V, Biochem biophys res commun 28 (1967) 815.

33. Stephens, R E, Quar~ rev biophys 1 4 (1969) 377. 34. Tanford, C & Lovrien, R, J Am chem soc 84

(1962) 1892. 35. Thompson, C M & Wolpert, L, Exptl cell res 32

(1963) 156. 36. Trevelyan, W E & Harrison, J S, Biochem j 50

(1952) 298. 37. Wohlfarth-Bottermann, K E, Int rev cytol 16

(1964) 61. 38. Wohlfarth-Bottermann, K E & Bhowmick, D K,

Exptl cell res 40 (1965) 252. 39. Wohlman, A & Allen, R D, J cell sci 3 (1968)

105. 40. Wolpert, L, Symp soc gen microbiol 15 (1965)

270. 41. Wolpert, L, Thompson, C M & O'Neill, C H,

Primitive motile systems (ed R D Allen & N Kamiya) p. 143. Academic Press, New York (1965).

Received July 9, 1970. Revised version received September 12, 1970

Exptl Cell Res 65

Documents

Microfilaments from amoeba proteins