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Molecular weight of base plates of bacteriophage T4D
Biochimica et Biophysica Acta, 386 (1975) 369-372
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 37007 M O L E C U L A R W E I G H T OF BASE PLATES OF BACTERIOPHAGE T4D
R. A. SULTANOVA, V. YA. CHERNYAK and B. F. POGLAZOV Laboratory of Bioorganic Chemistry, Moscow State University, Moscow (U.S.S.R.) (Received October 7th, 1974)
SUMMARY Highly purified base plates of bacteriophage T4D were obtained from lysate of gene 19 am mutant of this phage by differential centrifugation and sucrose gradient. Base plates were studied by means of high speed sedimentation equilibrium. The molecular weight determined by this method is (6.7 4- 0.2). 106.
Base plates constitute the central element of the tail of T-even bacteriophages. Cummings et al. [1] were the first to describe the method of isolating base plates of bacteriophage T4 and investigate their properties. Later is was demonstrated that base plates have a complex protein composition [2, 3] and contained approximately twelve different proteins; genes controlling their synthesis were identified for eight of them [3]. Further study of the composition and organization of base plates made it necessary to measure their molecular weight. This was the purpose of the present investigation. Base plates were produced with the aid of the amEl137 mutant of the T4D bacteriophage damaged in gene 19, which was kindly provided by Dr Wood. The mutant was grown on Escherichia coli B at 37 °C. In its turn E. coli B was grown on the synthetic medium (7 g Na2HPO+, 3 g KH2PO+, 1 g NH4C1, 0.5 g NaC1, 2 g glucose, 0.12 g MgSO+, 40 ml casein hydrolyzate per 1 1 of the medium) with intensive aeration in a 100-1 fermenter to bring the cell concentration to 2-10 a cells per ml. Then the culture was inf0cted with the phage with a multiplicity of 5 and 10 min later superinfected with the same amount of the phage. After that the cells were incubated for another 45 min. The bacterial mass was separated in the separator and resuspended in 1.5-2 1 of the culture liquid. The cells were lyzed with chloroform and left overnight at 4 °C. The resulting lyzate was supplemented with deoxyribonuclease (40 #g/ml) in the presence of Mg 2+ and ribonuclease (100 #g/ml) and kept for 2 h at 37 °C. Fragments of bacterial cells were separated by centrifugation at 6000 × g for 30 rain. Fragments of phage particles remaining in the supernatant were sedimented at 78 000 × g for 7 h. The pellet was suspended in 0.01 M Tris. HC1 buffer, pH 7.4. This mixture of various phage structures, capsids, polysheaths, base plates, fibers, was used for isolation and purification of base plates. The first purification was carried out in the 5-20?/o sucrose gradient in the SW27 rotor at 130 000 × g for 2.5 h at 2 °C. Further purifications were performed in the 10--45 ~o sucrose gradient in the SW39 rotor at 36 000 rev./min for 1 h and 15 min. The separated layers of base plates were sedi-
370 mented at 165 000 × g for 7 h. The pellets were suspended in 0.01 M Tris. HC1 buffer, pH 7.4. The preparation was tested for purity by means of electron microscopy and analytical centrifugation. Electron miscroscopy was carried out in a Hitachi-12 microscope with a magnification of 50 000. The preparations were contrasted with 2 % sodium phosphotungstate and placed onto the Formwar film support. The velocity centrifugation was carried out immediately before and after the equilibrium experiments, at 16 000 rev./min at 4and 20 °C in the six-channel cell in a Beckman model E ultracentrifuge equipped with scanning optics, scanning done at a minimal velocity and recording, at a velocity of 5 mm/s. After the last run the cell was opened, washed with buffer and a blank experiment with buffer was carried out in an ultracentrifuge equipped with interference optics. Electron microscope examination and velocity sedimentation of the samples showed we were dealing with homogeneous monomeric forms of base plates. The sedimentation coefficient was always 69 -+- 3 S, that is close to 77 S and 72 S demonstrated for monomers by Cummings et al. [1] and King [4] (for dimers these authors give the values of 124 S and 105 S). The equilibrium sedimentation was done in an ultracentrifuge equipped with interference optics, which was adjusted according to Richards et al. [5, 6] but with the interference mask being aligned from outside (Chernyak, V. Ya., unpublished). The camera lens was focused at the level of two-thirds of the height of the cell with sapphire windows. The sedimentation equilibrium experiments were performed by the method of Yphantis [7] in a six-channel 12 mm cell and in a An-J rotor at 2531 and 2809 rev./min (with overspeeding at 4609 and 4327 rev./min, respectively chosen on the basis of the equation of Hexner et al. [8]) and 4 °C. The artificial bottom was made of oil FC-43. In two experiments five solutions with initial concentrations of 0.23-0.62 mg/ml were tested. The experiments lasted 48 and 49 h. The equilibrium was considered to be achieved when the position of fringes near the bottom varied less than ±5/~m for 8 h. The velocity was measured with the aid of the odometer. Photography was made on plates Kodak II-G. The deflections of fringes were measured by means of the UIM-23 two-coordinate microscope (U.S.S.R.) (scale division, 1/zm) at 30-fold magnification. Deflections exceeding 90/~m with respect to the horizontal part of the fringes were used in calculations. Deflections of three fringes average were corrected with the aid of a blank. The molecular weight was calculated according to the equation:
2RT Mw,~ = ( 1 -
~O)'co2
dlny dr 2
where Mw,c is the apparent weight average molecular weight at concentration C; y, deflections of fringes; r, distance from the center of rotation; V,, partial specific volume; ¢, density of the solution; R, gas constant; T, absolute temperature; co, angular velocity. The partial specific volume was calculated on the basis of the amino acid composition of base plates (I) and corrected for 4 °C with the assumption that AI~V/dT= 5- 10-4 cmS/g per degree [9]. The calculations yielded V = 0.73 cm3/g. To determine the dependence of Mw,c on concentration, the experimental data ([ny, r 2) were linearly approximated for every five points by least-squares curve fitting.
371 The concentration corresponding to the central point was calculated according to the equation: "Y a(dn/c)d
C (mg/ml) =
---- 0.88 y ( r a m )
where ;t = 546.10 -7 cm, a = 1.2 cm (the width of the cell), An/c = 19.10-5"ml/mg protein refraction index increment and d = 272.10 -4 cm, the distance between fringes. Fig. 1 shows a typical plot o f l n y versus r 2 and Fig. 2 represents the corresponding Mw,c versus C plot.
Bottom
O5 4ny(mm) 0 -O5 -1.0 OO
-1.5
OO
Mwe 70
Ii
-2~
)gl
OQ I
°
o o
•
•
*
•
6.
-2Z F
I
5Q5
i
I
i
510 t-2 (cm2)
I
51,5
I
-~
I
I
I
I
I
I
J
I
I
0.1 0.2 0.3 0.4 0 5 0.6 0.7 0.8 0 9
)
-_~
1
C{mg I.ml)
Fig. 1. A typical plot of lny versus r ~ in sedimentation equilibrium experiments performed with the base plates of the T4D bacteriophage. The solution had Co = 0.62 mg/ml and was centrifuged 48 h (including overspeeding) at 2785 rev./min; temperature 4 °C; y, deflections of fringes in ram; r, distance from the centre of rotation in cm. Fig. 2. A typical plot of Mw,c versus C (in the experiment shown in Fig. 1). Concentration C in mg/ml.
For each of the five solutions tested mean-square deviations of Mw,~ from the mean value did not exceed zkl.5 %. The molecular weight of base plates Mw,c derived from five measurements was (6.7 4- 0.2)- 106. The electron microscopy and velocity sedimentation are proving this value to be the molecular weight of base plate monomers. ACKNOWLEDGEMENT
We express our sincere gratitude to Dr Wood for the amEI137 mutant of the T4 bacteriophage he has kindly supplied to us. REFERENCES 1 Cummings, D. J., Kusy, A. K., Chapman, V. A., De Long, S. S. and Stone, K. R. (1970) J. Virol. 6, 545-555
372 2 3 4 5 6 7 8 9
Poglazov, B. F., Rodikova, L. P. and Sultanova, R. A. (1972) J. Virol. 10, 810-815 King, J. and Mykolajewycz, N. (1973) J. Mol. Biol. 75, 339-358 King, J. (1971) J. Mol. Biol. 58, 693-709 Richards, E. G., Teller, D. and Schachman, H. R. (1971) Anal. Biochem. 41, 189-214 Richards, E. G., Teller, D. and Schachman, H. R. (1971) Anal. Biochem. 41,215-247 Yphantis, D. A. (1964) Biochemistry 3, 297-317 Hexner, P. E., Radford, L. E. and Beams, J. W. (1961) Proc. Natl. Acad. Sci. U.S. 47, 1848-1852 Svedberg, The. and Pedersen, K. O. (1940) The Ultracentrifuge, Oxford
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 37007 M O L E C U L A R W E I G H T OF BASE PLATES OF BACTERIOPHAGE T4D
R. A. SULTANOVA, V. YA. CHERNYAK and B. F. POGLAZOV Laboratory of Bioorganic Chemistry, Moscow State University, Moscow (U.S.S.R.) (Received October 7th, 1974)
SUMMARY Highly purified base plates of bacteriophage T4D were obtained from lysate of gene 19 am mutant of this phage by differential centrifugation and sucrose gradient. Base plates were studied by means of high speed sedimentation equilibrium. The molecular weight determined by this method is (6.7 4- 0.2). 106.
Base plates constitute the central element of the tail of T-even bacteriophages. Cummings et al. [1] were the first to describe the method of isolating base plates of bacteriophage T4 and investigate their properties. Later is was demonstrated that base plates have a complex protein composition [2, 3] and contained approximately twelve different proteins; genes controlling their synthesis were identified for eight of them [3]. Further study of the composition and organization of base plates made it necessary to measure their molecular weight. This was the purpose of the present investigation. Base plates were produced with the aid of the amEl137 mutant of the T4D bacteriophage damaged in gene 19, which was kindly provided by Dr Wood. The mutant was grown on Escherichia coli B at 37 °C. In its turn E. coli B was grown on the synthetic medium (7 g Na2HPO+, 3 g KH2PO+, 1 g NH4C1, 0.5 g NaC1, 2 g glucose, 0.12 g MgSO+, 40 ml casein hydrolyzate per 1 1 of the medium) with intensive aeration in a 100-1 fermenter to bring the cell concentration to 2-10 a cells per ml. Then the culture was inf0cted with the phage with a multiplicity of 5 and 10 min later superinfected with the same amount of the phage. After that the cells were incubated for another 45 min. The bacterial mass was separated in the separator and resuspended in 1.5-2 1 of the culture liquid. The cells were lyzed with chloroform and left overnight at 4 °C. The resulting lyzate was supplemented with deoxyribonuclease (40 #g/ml) in the presence of Mg 2+ and ribonuclease (100 #g/ml) and kept for 2 h at 37 °C. Fragments of bacterial cells were separated by centrifugation at 6000 × g for 30 rain. Fragments of phage particles remaining in the supernatant were sedimented at 78 000 × g for 7 h. The pellet was suspended in 0.01 M Tris. HC1 buffer, pH 7.4. This mixture of various phage structures, capsids, polysheaths, base plates, fibers, was used for isolation and purification of base plates. The first purification was carried out in the 5-20?/o sucrose gradient in the SW27 rotor at 130 000 × g for 2.5 h at 2 °C. Further purifications were performed in the 10--45 ~o sucrose gradient in the SW39 rotor at 36 000 rev./min for 1 h and 15 min. The separated layers of base plates were sedi-
370 mented at 165 000 × g for 7 h. The pellets were suspended in 0.01 M Tris. HC1 buffer, pH 7.4. The preparation was tested for purity by means of electron microscopy and analytical centrifugation. Electron miscroscopy was carried out in a Hitachi-12 microscope with a magnification of 50 000. The preparations were contrasted with 2 % sodium phosphotungstate and placed onto the Formwar film support. The velocity centrifugation was carried out immediately before and after the equilibrium experiments, at 16 000 rev./min at 4and 20 °C in the six-channel cell in a Beckman model E ultracentrifuge equipped with scanning optics, scanning done at a minimal velocity and recording, at a velocity of 5 mm/s. After the last run the cell was opened, washed with buffer and a blank experiment with buffer was carried out in an ultracentrifuge equipped with interference optics. Electron microscope examination and velocity sedimentation of the samples showed we were dealing with homogeneous monomeric forms of base plates. The sedimentation coefficient was always 69 -+- 3 S, that is close to 77 S and 72 S demonstrated for monomers by Cummings et al. [1] and King [4] (for dimers these authors give the values of 124 S and 105 S). The equilibrium sedimentation was done in an ultracentrifuge equipped with interference optics, which was adjusted according to Richards et al. [5, 6] but with the interference mask being aligned from outside (Chernyak, V. Ya., unpublished). The camera lens was focused at the level of two-thirds of the height of the cell with sapphire windows. The sedimentation equilibrium experiments were performed by the method of Yphantis [7] in a six-channel 12 mm cell and in a An-J rotor at 2531 and 2809 rev./min (with overspeeding at 4609 and 4327 rev./min, respectively chosen on the basis of the equation of Hexner et al. [8]) and 4 °C. The artificial bottom was made of oil FC-43. In two experiments five solutions with initial concentrations of 0.23-0.62 mg/ml were tested. The experiments lasted 48 and 49 h. The equilibrium was considered to be achieved when the position of fringes near the bottom varied less than ±5/~m for 8 h. The velocity was measured with the aid of the odometer. Photography was made on plates Kodak II-G. The deflections of fringes were measured by means of the UIM-23 two-coordinate microscope (U.S.S.R.) (scale division, 1/zm) at 30-fold magnification. Deflections exceeding 90/~m with respect to the horizontal part of the fringes were used in calculations. Deflections of three fringes average were corrected with the aid of a blank. The molecular weight was calculated according to the equation:
2RT Mw,~ = ( 1 -
~O)'co2
dlny dr 2
where Mw,c is the apparent weight average molecular weight at concentration C; y, deflections of fringes; r, distance from the center of rotation; V,, partial specific volume; ¢, density of the solution; R, gas constant; T, absolute temperature; co, angular velocity. The partial specific volume was calculated on the basis of the amino acid composition of base plates (I) and corrected for 4 °C with the assumption that AI~V/dT= 5- 10-4 cmS/g per degree [9]. The calculations yielded V = 0.73 cm3/g. To determine the dependence of Mw,c on concentration, the experimental data ([ny, r 2) were linearly approximated for every five points by least-squares curve fitting.
371 The concentration corresponding to the central point was calculated according to the equation: "Y a(dn/c)d
C (mg/ml) =
---- 0.88 y ( r a m )
where ;t = 546.10 -7 cm, a = 1.2 cm (the width of the cell), An/c = 19.10-5"ml/mg protein refraction index increment and d = 272.10 -4 cm, the distance between fringes. Fig. 1 shows a typical plot o f l n y versus r 2 and Fig. 2 represents the corresponding Mw,c versus C plot.
Bottom
O5 4ny(mm) 0 -O5 -1.0 OO
-1.5
OO
Mwe 70
Ii
-2~
)gl
OQ I
°
o o
•
•
*
•
6.
-2Z F
I
5Q5
i
I
i
510 t-2 (cm2)
I
51,5
I
-~
I
I
I
I
I
I
J
I
I
0.1 0.2 0.3 0.4 0 5 0.6 0.7 0.8 0 9
)
-_~
1
C{mg I.ml)
Fig. 1. A typical plot of lny versus r ~ in sedimentation equilibrium experiments performed with the base plates of the T4D bacteriophage. The solution had Co = 0.62 mg/ml and was centrifuged 48 h (including overspeeding) at 2785 rev./min; temperature 4 °C; y, deflections of fringes in ram; r, distance from the centre of rotation in cm. Fig. 2. A typical plot of Mw,c versus C (in the experiment shown in Fig. 1). Concentration C in mg/ml.
For each of the five solutions tested mean-square deviations of Mw,~ from the mean value did not exceed zkl.5 %. The molecular weight of base plates Mw,c derived from five measurements was (6.7 4- 0.2)- 106. The electron microscopy and velocity sedimentation are proving this value to be the molecular weight of base plate monomers. ACKNOWLEDGEMENT
We express our sincere gratitude to Dr Wood for the amEI137 mutant of the T4 bacteriophage he has kindly supplied to us. REFERENCES 1 Cummings, D. J., Kusy, A. K., Chapman, V. A., De Long, S. S. and Stone, K. R. (1970) J. Virol. 6, 545-555
372 2 3 4 5 6 7 8 9
Poglazov, B. F., Rodikova, L. P. and Sultanova, R. A. (1972) J. Virol. 10, 810-815 King, J. and Mykolajewycz, N. (1973) J. Mol. Biol. 75, 339-358 King, J. (1971) J. Mol. Biol. 58, 693-709 Richards, E. G., Teller, D. and Schachman, H. R. (1971) Anal. Biochem. 41, 189-214 Richards, E. G., Teller, D. and Schachman, H. R. (1971) Anal. Biochem. 41,215-247 Yphantis, D. A. (1964) Biochemistry 3, 297-317 Hexner, P. E., Radford, L. E. and Beams, J. W. (1961) Proc. Natl. Acad. Sci. U.S. 47, 1848-1852 Svedberg, The. and Pedersen, K. O. (1940) The Ultracentrifuge, Oxford