JOURNAL
OF INVERTEBRATE
PATHOLOGY
25,97-101
(197%
Proteolytic Cleavage of Polyhedral Protein during Dissolution of Inclusion Bodies of the Nuclear Polyhedrosis Viruses of Bombyx mori and Galieria me//one/la under Alkaline Conditions E. A. KOZLOV, Institute
of
Molecular
Biology
X. and
M.
SIDOROVA,
Genetics, Kiev, Received
AND
Academy U.S.S.R. August
S.
B.
of Sciences
SEREBRYANI of
the
Ukrainian
S.S.R.,
87, 1973
Inclusion bodies (IB) of nuclear polyhedrosis viruses (NPV) of Bombyx mori and GalZeria mellonella were dissolved in 67% acetic acid and in sodium carbonate solution at pH 11.0. The polyhedral protein preparations obtained in this way were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by ultracentrifugation. The polyhedral proteins of B. mori and G. mellonella NPV obtained by dissolving IB in acetic acid were shown to have mainly one component with a molecular weight of about -t28,000 and sedimentation coefficients in 0.1 M NaOH of 1.8 and 2.1 S, respectively. During dissolution of IB in alkaline condition, both the proteins are cleaved and reveal several components. The quantity and the ratio of the components depend on the duration of storing and on the temperature during the dissolution of IB and on the stage of insect development at the time of IB isolation. It is suggested that the cleavage of IB protein is a result of alkaline proteinase(s) activity.
To obtain polyhedral protein (PP), Bergold (1947) proposed dissolving IB in a solution of sodium carbonate. He reached the conclusion that highly-homogeneous PP obtained in this way dissociates to minimal (“second”) subunits only in 0.1-0.5 M NaOH. The “second” subunit of PP of R. mori NPV was shown by him to have the sedimentation constant 1.49 S and a molecular weight of about, 20,000. However, acrording to our data the minimal subunjt of PP of B. W.OT~ NPV ha!: in 0.1 11 NaOH a sediment.ation coefficient of 1.1 S and a molecular weight of about, 11,000 iliozlov et al., 1969). Recently, reports have appeared which indicate that IB proteins of the different insect viruses have several protein components (Harrap, 1972; Longworth et al., 1972).
We applied the molecular weight analysis by electrophoresis in polyacrylamide gel with sodium dodcryl sulfate in order to test PP homogcncity and to elucidate the discrepancies of molecular weights reported by different investigators. The results of this investigat,ion are described in this paper. M.~TERIALs
Inclusion
Rodits
ANL, (113)
Press, Inc. All rights
of R. mori
Tht NP1’ n-c’rc’ isol:lt~tl 1)~ rcpcntcd crntrifugation of cadavers of silkworm larvae that had died from nuclear polyhedrosis f Kozlov et al.. 1966). Inclusion bodies of C. mellonclln NW isolated from larvae infected I)!; SW were donated by Dr. E. -1;. Zherehtsorn. Thth air-dried IB had been stored for 2-3 yr at cold temperature in a bottle with ground-in stopper (“old” IB).
97 Copyright o 1975 by Academic Printed in the United States.
METHODS
of reproduction
in any form
reserved.
98 Polyhedral
KOZLOV,
Protein
SIDOROVA
(PP)
We prepared three PP preparations. In the case of preparation 1 (PP-l), 10 mg of IB were dissolved in 20 ml of 67% acetic acid for 2 hr at 8OC. The residue was centrifuged for 1 hr at 50,OOOg at 4OC. The residue recovery was lO-15%. The supernatant was diluted 10 times with water and lyophilized. In the case of preparation 2 (PP-2)) 10 mg of IB were dissolved in 20 ml of a solution of 0.05 M Na,CO, plus 0.1 M NaCl at pH 11.0 for 2 hr at room temperature or 37OC. The residue was centrifuged for 1 hr at 35,000g at 4OC. The residue recovery was 5-10s. The supernatant was subjected to dialysis against water-ammonia at pH 7-8 and lyophilized. In the case of preparation 3 (PP-3), 10 mg of IB were dissolved completely as PP-2 for 5 min at 90°C. The solution was cooled quickly and then processed as in the case of PP-2. Sodium Dodecyl Sulfate Electrophoresis in Polyacrylamide Gel (SDS-PAGE) The acrylamide gels employed contained 10% acrylamide, 0.2% N,N’-methylenebisacrylamide, 0.6% N,N,N’,N’-tetramethylethylenediamine, 0.07% ammonium persulfate, 0.5 M urea, 0.1% SDS, 0.1% p-mercaptoethanol (ME), and 0.01 M sodium phosphate buffer at pH 7.4. The preparation of gel and of the samples was made by the methods described by Shapiro et al. (1967) and Weber and Osborn (1969). Two-hundredths of a milliliter samples containing 50-100 pg of protein were applied to the gel. Electrophoresis was carried out at a current of 2 mA/gel for 2 hr. After electrophoresis the gel was immersed for 18 hr in a 20% solution of sulphosalicylic acid and stained with lo/O amido black B in 7% acetic acid. The molecular weight of PP components was estimated by using the following calibration proteins as standards: bovine serum albumine (BSA), MW 68,000; pepsin, MW 35,000; pancreatic ribonuclease, MW 14,000.
AND
SEREBRYANI
Ultracentrifugation Sedimentation experiments were performed in a MOM Model G-120 analytical ultracentrifuge. The PP were dissolved in a 0.1 M solution of NaOH and dialyzed against the same for 2 hr. The protein concentration was 0.7%. RESULTS
AND
SDS-Polyacrylamide
DISCUSSION
Gel Electrophoresis
It is known that different chemical alterations arise after alkaline treatment of proteins (Bohak, 1964). Kavsan et al. (1970) have proposed for disso1vin.gIB of B. mori NPV the use of the method described by Fraenkel-Conrat (1957). We applied this method with a slight modification. Polyhedral protein obtained by dissolving in 67% acetic acid (PP-1) have revealed by SDS-PAGE a single component having a molecular weight of about 28,000 (Fig. la). Inclusion bodies dissolved in 0.01 M sodium phosphate buffer at pH 7.4 containing 2% SDS, lo/O ME, and 0.5 M urea for 5 min at 100°C and then for 2 hr at 37OC revealed by SDS-PAGE mainly one component with a molecular weight of about 28,000 (Fig. lb). Inspection of the gel has revealed the presence of minor components with higher and lower molecular weights. The minor component with a higher molecular weight is possibly an aggregate of a major component (MW 28,000). However, PP-2 obtained under alkaline conditions at room temperature contains a major component (MW 11,00&14,000) and a minor one (MW 23,000) (Fig. lc). The quantity and ratio of PP-2 components depend on the time of storage, the dissolution temperature of IB, and the stage of insect development. PP-2 obtained by dissolving “old” IB of B. mori at room temperature contains five components (MW 28,000,23,000, 19,000, 14,000, and 11,000) (Fig. Id). PP-2 obtained by dissolving the same “old” IB at 37OC revealed a diffusion band with a molecular weight ranging from 11,000 to 15,000 (Fig. le).
PROTEOLYTIC
CLEAVAGE
IN
B. mori
AND
G.
99
mellonella
FIG. 1. Electrophoretic patterns of inclusion body protein preparations in polyacrylamide gel containing sodium dodecyl sulfate. Preparation characteristics are described in the text. a, b, c, 11, and i = patterns of the proteins of B. mori and G. mellonella NPV inclusion bodies (identical for both proteins) ; d, e, and g = patterns of the proteins of B. mori NPV NPV inclusion body; j and inclusion body; f = pattern of the protein of G. mellonella k = pat,terns of bovine serum albumine. Migration is from top to bottom. PP-2
obtained
by dissolving
“old”
IB
of
G. mellonella at room temperature or at 37% contains a major component (MW 28,000) and minor one (MW 25,000) (Fig. If). When we obtained PP-2 by dissolving fresh IB newly isolated from B. m,ori
pupae, the protein contained two major components with molecular weights of 28000 and 23,000, respectively. In addition, there are two minor components with molecular weights of 19,000 and 14,000, respectively (Fig. Ig) . It may be supposedthat all these component.s originate as a result of cleavage of the componet with a molecuiar weight of 28.000 by activity of an alkaline protease. Yamafugi et al. (1960) showed that IB of K. 021~; NPV contains an alkaline protease. This is confirmed by the fact that PP-3 incubated for 2 hr at’ 37°C and pH 11.0 is not cleaved and contains the component with a molecular weight of 28,000 (Fig. is lh) It may be supposed that protease inactivated during IB dissolution at 90% (see the preparation of PP-3 under Materials and Methods). To determine the presence of protease in IB of G. mellonella NPV, we incubated 10
mg of PP-3 and 10 mg of BSA with 2 mg of G. mellonella IB for 2 hr at 37OC and pH 11.0. During incubation, the IB were dissolved. Both PP-3 and BSA were cleaved (Figs. li and li, respectively). An electrophoregram of BSA incubated under the same conditions without IB is presented in Fig. lk. It can be seen that BSA is not cleaved during incubation at 37OC and pH 11.0 without IB. It is obvious that cleavage PP-3 and BSA during incubation at 37OC and pH 11.0 depend on the presence of protease activity in G. ruellon.ella IB. One may suppose that Protease
activity
can
arise
from
contami-
nants in IB preparations, which may increase with storage, and not, be an enzyme from thc~ IB themselves. It is noted, however, that the IB were treated during isolation by pancrcatin and carefully cleaned by repeated centrifugation (Kozlov et al., 1966). From the results shown in Figs. Id, le, and If. it appears that the protein during dissolution of “old” IB is cleaved to some extent, especially in the case of G. wellonella “old” IB (Fig. If). It is clear that alkaline proteasc activity is decreased with storage.
KOZLOV,
100
SIDOROVA
FIG. 2. Sedimentation patterns of the e) and G. mellonella NPV (a and d). text. The pictures were taken after 45 62 min at 50,120 rpm (c), 54 min at 54,530
Ultracentrifugafion
AND SEREBRYANI
inclusion body proteins of B. mori NPV (b, c, and For the characteristics of ,the preparations see the min at 53,040 rpm (a), 54 min at 54,540 rpm (b), rpm (d), and 29 min at 59,900 rpm (e).
tions the presence of a major component of B. mori PP of approximately 11-12 S. These authors have postulated that a minimal subunit of PP with a molecular weight of 20,000 and a sedimentation coefficient of 1.5 S (Bergold, 1947) or 11,000 and 1.1 S (Kozlov et al., 1969) aggregates to form higher molecular weight components. The data described here may lead to the supposition that the IB of B. mori and G. mellonella NPV are built mainly from a protein with a molecular weight of 28,000 and a sedimentation coefficient of about 2.0 S. During the treatment of IB in a sodium carbonate solution the protein evidently is cleaved by protease from the IB themselves. It seems possible that an alkaline protease is also present in IB of other insect NPV.
The cleavage of PP during dissolution of IB under alkaline conditions was confirmed by ultracentrifugation. PP-1 of G. mellonella and B. mori NPV sedimented in 0.1 it4 NaOH with sedimentation coefficients of 2.1 and 1.8 S, respectively (Figs. 2a, 2b). PP-2 obtained from newly isolated IB of B. mori NPV sediments with a sedimentation coefficient 1.1 S (Fig. 2~). This result coincides with our data published earlier (Kozlov et al., 1969). The analytical ultracentrifuge pattern of PP-2 of G. mellonella NPV obtained from newly isolated IB is shown in Fig. 2d. A broad 2.2-S peak is seen with a slow-sedimenting peak at a sedimentation coefficient of about 1 S. When PP-2 of B. mori NPV containing five components (Fig. Id) was subjected to ultracentrifugation, a spreading boundary was formed ACKNOWLEDGMENTS having a mean sedimentation coefficient of We wish to thank Professor S. M. Gershenson 1.5 S (Fig. 2e). This result coincides with for critical remarks, V. Vember for performing data on the “second” PP subunit published analytical centrifugation, and G. Dzvinchuk for by Bergold (1947). technical assistance. It seemspossible that the sedimentation coefficients of the minimal subunits of the REFERENCES polyhedral protein of NPV of different in- BERGOLD, C. H. 1947. Die Isolierung des Polyedersects can differ because of the various ratios Virus und die Natur der Polyeder. 2. Nalwforsch., B 2, 122-143. of the components in PP preparations obBOHAK, 2. 1964. N-(on-2-Amino-2-carboxytained by dissolving IB under alkaline ethyl)-L-lysine, a new amino acid formed on conditions. alkaline treatment of proteins. J. Biol. Chem., Bergold (1947) and Kozlov et al. (1969) 239, 2878-2887. observed under the proper alkaline condi- FRAENKEL-CONRAT, H. 1957. Degradation of to-
PROTEOLYTIC
CLEAVAGE
IN
bacco mosaic virus with acetic acid. T’irology, 4, l-4. HARRAY. K. A. 1972. The structure of nuclear polyhedrosis viruses. I. The inclusion body. l.iroZogy. 50, 114-123. KAYBAN,
V.
M.,
&TSMAN,
M.
S., AND
SEREBRYANI,
S. B. 1970. Obtaining of polyhedral protein of Borrelinavirus bombycis by treatment of polyhedra with acetic acid. ,Wikrobiol. Zh., 32, 355-359. (In Ukraini:m wit 11 Russian and English summaries.) K~ZLOV, E. A., SOGULJAEVA,V. M., AND SEREBRYANI. S. B. 1966. Invratigntion of the primary structurp of polyhedral prokin of thr nuclear polylrcdrosis virus of silkworm. C’kr. Khim. Zh.. 32, 875-878. (In Russian.) Bozr>ov, E. A., SOGULJAEV.4. V. M., LEVITINA, T. I,., VERESCHAK, V.: AKD SERRBRYAXI, S, R. 1969. Purification of polyhedral protein of nuclear polyhedrosis virus of silkworm and the pro-
R.
WlO?+i
AND
G.
WdOndhJ
101
cess of its association in solution. Biokhimiyn, 34, 679-685. (In Russian with English summary’.) LoxGWOIWII, .J. F.. ROBERTSON, J. S., AND PAYNE:, C. c’. 1972. Thr lkfication :rnd properties of inclusion body protck of thr granulosis virus of Pieris 00/ssicfle. J. Ztl~lrrlebr. F’crthol., 19, 42-50.
I
OF INVERTEBRATE
PATHOLOGY
25,97-101
(197%
Proteolytic Cleavage of Polyhedral Protein during Dissolution of Inclusion Bodies of the Nuclear Polyhedrosis Viruses of Bombyx mori and Galieria me//one/la under Alkaline Conditions E. A. KOZLOV, Institute
of
Molecular
Biology
X. and
M.
SIDOROVA,
Genetics, Kiev, Received
AND
Academy U.S.S.R. August
S.
B.
of Sciences
SEREBRYANI of
the
Ukrainian
S.S.R.,
87, 1973
Inclusion bodies (IB) of nuclear polyhedrosis viruses (NPV) of Bombyx mori and GalZeria mellonella were dissolved in 67% acetic acid and in sodium carbonate solution at pH 11.0. The polyhedral protein preparations obtained in this way were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by ultracentrifugation. The polyhedral proteins of B. mori and G. mellonella NPV obtained by dissolving IB in acetic acid were shown to have mainly one component with a molecular weight of about -t28,000 and sedimentation coefficients in 0.1 M NaOH of 1.8 and 2.1 S, respectively. During dissolution of IB in alkaline condition, both the proteins are cleaved and reveal several components. The quantity and the ratio of the components depend on the duration of storing and on the temperature during the dissolution of IB and on the stage of insect development at the time of IB isolation. It is suggested that the cleavage of IB protein is a result of alkaline proteinase(s) activity.
To obtain polyhedral protein (PP), Bergold (1947) proposed dissolving IB in a solution of sodium carbonate. He reached the conclusion that highly-homogeneous PP obtained in this way dissociates to minimal (“second”) subunits only in 0.1-0.5 M NaOH. The “second” subunit of PP of R. mori NPV was shown by him to have the sedimentation constant 1.49 S and a molecular weight of about, 20,000. However, acrording to our data the minimal subunjt of PP of B. W.OT~ NPV ha!: in 0.1 11 NaOH a sediment.ation coefficient of 1.1 S and a molecular weight of about, 11,000 iliozlov et al., 1969). Recently, reports have appeared which indicate that IB proteins of the different insect viruses have several protein components (Harrap, 1972; Longworth et al., 1972).
We applied the molecular weight analysis by electrophoresis in polyacrylamide gel with sodium dodcryl sulfate in order to test PP homogcncity and to elucidate the discrepancies of molecular weights reported by different investigators. The results of this investigat,ion are described in this paper. M.~TERIALs
Inclusion
Rodits
ANL, (113)
Press, Inc. All rights
of R. mori
Tht NP1’ n-c’rc’ isol:lt~tl 1)~ rcpcntcd crntrifugation of cadavers of silkworm larvae that had died from nuclear polyhedrosis f Kozlov et al.. 1966). Inclusion bodies of C. mellonclln NW isolated from larvae infected I)!; SW were donated by Dr. E. -1;. Zherehtsorn. Thth air-dried IB had been stored for 2-3 yr at cold temperature in a bottle with ground-in stopper (“old” IB).
97 Copyright o 1975 by Academic Printed in the United States.
METHODS
of reproduction
in any form
reserved.
98 Polyhedral
KOZLOV,
Protein
SIDOROVA
(PP)
We prepared three PP preparations. In the case of preparation 1 (PP-l), 10 mg of IB were dissolved in 20 ml of 67% acetic acid for 2 hr at 8OC. The residue was centrifuged for 1 hr at 50,OOOg at 4OC. The residue recovery was lO-15%. The supernatant was diluted 10 times with water and lyophilized. In the case of preparation 2 (PP-2)) 10 mg of IB were dissolved in 20 ml of a solution of 0.05 M Na,CO, plus 0.1 M NaCl at pH 11.0 for 2 hr at room temperature or 37OC. The residue was centrifuged for 1 hr at 35,000g at 4OC. The residue recovery was 5-10s. The supernatant was subjected to dialysis against water-ammonia at pH 7-8 and lyophilized. In the case of preparation 3 (PP-3), 10 mg of IB were dissolved completely as PP-2 for 5 min at 90°C. The solution was cooled quickly and then processed as in the case of PP-2. Sodium Dodecyl Sulfate Electrophoresis in Polyacrylamide Gel (SDS-PAGE) The acrylamide gels employed contained 10% acrylamide, 0.2% N,N’-methylenebisacrylamide, 0.6% N,N,N’,N’-tetramethylethylenediamine, 0.07% ammonium persulfate, 0.5 M urea, 0.1% SDS, 0.1% p-mercaptoethanol (ME), and 0.01 M sodium phosphate buffer at pH 7.4. The preparation of gel and of the samples was made by the methods described by Shapiro et al. (1967) and Weber and Osborn (1969). Two-hundredths of a milliliter samples containing 50-100 pg of protein were applied to the gel. Electrophoresis was carried out at a current of 2 mA/gel for 2 hr. After electrophoresis the gel was immersed for 18 hr in a 20% solution of sulphosalicylic acid and stained with lo/O amido black B in 7% acetic acid. The molecular weight of PP components was estimated by using the following calibration proteins as standards: bovine serum albumine (BSA), MW 68,000; pepsin, MW 35,000; pancreatic ribonuclease, MW 14,000.
AND
SEREBRYANI
Ultracentrifugation Sedimentation experiments were performed in a MOM Model G-120 analytical ultracentrifuge. The PP were dissolved in a 0.1 M solution of NaOH and dialyzed against the same for 2 hr. The protein concentration was 0.7%. RESULTS
AND
SDS-Polyacrylamide
DISCUSSION
Gel Electrophoresis
It is known that different chemical alterations arise after alkaline treatment of proteins (Bohak, 1964). Kavsan et al. (1970) have proposed for disso1vin.gIB of B. mori NPV the use of the method described by Fraenkel-Conrat (1957). We applied this method with a slight modification. Polyhedral protein obtained by dissolving in 67% acetic acid (PP-1) have revealed by SDS-PAGE a single component having a molecular weight of about 28,000 (Fig. la). Inclusion bodies dissolved in 0.01 M sodium phosphate buffer at pH 7.4 containing 2% SDS, lo/O ME, and 0.5 M urea for 5 min at 100°C and then for 2 hr at 37OC revealed by SDS-PAGE mainly one component with a molecular weight of about 28,000 (Fig. lb). Inspection of the gel has revealed the presence of minor components with higher and lower molecular weights. The minor component with a higher molecular weight is possibly an aggregate of a major component (MW 28,000). However, PP-2 obtained under alkaline conditions at room temperature contains a major component (MW 11,00&14,000) and a minor one (MW 23,000) (Fig. lc). The quantity and ratio of PP-2 components depend on the time of storage, the dissolution temperature of IB, and the stage of insect development. PP-2 obtained by dissolving “old” IB of B. mori at room temperature contains five components (MW 28,000,23,000, 19,000, 14,000, and 11,000) (Fig. Id). PP-2 obtained by dissolving the same “old” IB at 37OC revealed a diffusion band with a molecular weight ranging from 11,000 to 15,000 (Fig. le).
PROTEOLYTIC
CLEAVAGE
IN
B. mori
AND
G.
99
mellonella
FIG. 1. Electrophoretic patterns of inclusion body protein preparations in polyacrylamide gel containing sodium dodecyl sulfate. Preparation characteristics are described in the text. a, b, c, 11, and i = patterns of the proteins of B. mori and G. mellonella NPV inclusion bodies (identical for both proteins) ; d, e, and g = patterns of the proteins of B. mori NPV NPV inclusion body; j and inclusion body; f = pattern of the protein of G. mellonella k = pat,terns of bovine serum albumine. Migration is from top to bottom. PP-2
obtained
by dissolving
“old”
IB
of
G. mellonella at room temperature or at 37% contains a major component (MW 28,000) and minor one (MW 25,000) (Fig. If). When we obtained PP-2 by dissolving fresh IB newly isolated from B. m,ori
pupae, the protein contained two major components with molecular weights of 28000 and 23,000, respectively. In addition, there are two minor components with molecular weights of 19,000 and 14,000, respectively (Fig. Ig) . It may be supposedthat all these component.s originate as a result of cleavage of the componet with a molecuiar weight of 28.000 by activity of an alkaline protease. Yamafugi et al. (1960) showed that IB of K. 021~; NPV contains an alkaline protease. This is confirmed by the fact that PP-3 incubated for 2 hr at’ 37°C and pH 11.0 is not cleaved and contains the component with a molecular weight of 28,000 (Fig. is lh) It may be supposed that protease inactivated during IB dissolution at 90% (see the preparation of PP-3 under Materials and Methods). To determine the presence of protease in IB of G. mellonella NPV, we incubated 10
mg of PP-3 and 10 mg of BSA with 2 mg of G. mellonella IB for 2 hr at 37OC and pH 11.0. During incubation, the IB were dissolved. Both PP-3 and BSA were cleaved (Figs. li and li, respectively). An electrophoregram of BSA incubated under the same conditions without IB is presented in Fig. lk. It can be seen that BSA is not cleaved during incubation at 37OC and pH 11.0 without IB. It is obvious that cleavage PP-3 and BSA during incubation at 37OC and pH 11.0 depend on the presence of protease activity in G. ruellon.ella IB. One may suppose that Protease
activity
can
arise
from
contami-
nants in IB preparations, which may increase with storage, and not, be an enzyme from thc~ IB themselves. It is noted, however, that the IB were treated during isolation by pancrcatin and carefully cleaned by repeated centrifugation (Kozlov et al., 1966). From the results shown in Figs. Id, le, and If. it appears that the protein during dissolution of “old” IB is cleaved to some extent, especially in the case of G. wellonella “old” IB (Fig. If). It is clear that alkaline proteasc activity is decreased with storage.
KOZLOV,
100
SIDOROVA
FIG. 2. Sedimentation patterns of the e) and G. mellonella NPV (a and d). text. The pictures were taken after 45 62 min at 50,120 rpm (c), 54 min at 54,530
Ultracentrifugafion
AND SEREBRYANI
inclusion body proteins of B. mori NPV (b, c, and For the characteristics of ,the preparations see the min at 53,040 rpm (a), 54 min at 54,540 rpm (b), rpm (d), and 29 min at 59,900 rpm (e).
tions the presence of a major component of B. mori PP of approximately 11-12 S. These authors have postulated that a minimal subunit of PP with a molecular weight of 20,000 and a sedimentation coefficient of 1.5 S (Bergold, 1947) or 11,000 and 1.1 S (Kozlov et al., 1969) aggregates to form higher molecular weight components. The data described here may lead to the supposition that the IB of B. mori and G. mellonella NPV are built mainly from a protein with a molecular weight of 28,000 and a sedimentation coefficient of about 2.0 S. During the treatment of IB in a sodium carbonate solution the protein evidently is cleaved by protease from the IB themselves. It seems possible that an alkaline protease is also present in IB of other insect NPV.
The cleavage of PP during dissolution of IB under alkaline conditions was confirmed by ultracentrifugation. PP-1 of G. mellonella and B. mori NPV sedimented in 0.1 it4 NaOH with sedimentation coefficients of 2.1 and 1.8 S, respectively (Figs. 2a, 2b). PP-2 obtained from newly isolated IB of B. mori NPV sediments with a sedimentation coefficient 1.1 S (Fig. 2~). This result coincides with our data published earlier (Kozlov et al., 1969). The analytical ultracentrifuge pattern of PP-2 of G. mellonella NPV obtained from newly isolated IB is shown in Fig. 2d. A broad 2.2-S peak is seen with a slow-sedimenting peak at a sedimentation coefficient of about 1 S. When PP-2 of B. mori NPV containing five components (Fig. Id) was subjected to ultracentrifugation, a spreading boundary was formed ACKNOWLEDGMENTS having a mean sedimentation coefficient of We wish to thank Professor S. M. Gershenson 1.5 S (Fig. 2e). This result coincides with for critical remarks, V. Vember for performing data on the “second” PP subunit published analytical centrifugation, and G. Dzvinchuk for by Bergold (1947). technical assistance. It seemspossible that the sedimentation coefficients of the minimal subunits of the REFERENCES polyhedral protein of NPV of different in- BERGOLD, C. H. 1947. Die Isolierung des Polyedersects can differ because of the various ratios Virus und die Natur der Polyeder. 2. Nalwforsch., B 2, 122-143. of the components in PP preparations obBOHAK, 2. 1964. N-(on-2-Amino-2-carboxytained by dissolving IB under alkaline ethyl)-L-lysine, a new amino acid formed on conditions. alkaline treatment of proteins. J. Biol. Chem., Bergold (1947) and Kozlov et al. (1969) 239, 2878-2887. observed under the proper alkaline condi- FRAENKEL-CONRAT, H. 1957. Degradation of to-
PROTEOLYTIC
CLEAVAGE
IN
bacco mosaic virus with acetic acid. T’irology, 4, l-4. HARRAY. K. A. 1972. The structure of nuclear polyhedrosis viruses. I. The inclusion body. l.iroZogy. 50, 114-123. KAYBAN,
V.
M.,
&TSMAN,
M.
S., AND
SEREBRYANI,
S. B. 1970. Obtaining of polyhedral protein of Borrelinavirus bombycis by treatment of polyhedra with acetic acid. ,Wikrobiol. Zh., 32, 355-359. (In Ukraini:m wit 11 Russian and English summaries.) K~ZLOV, E. A., SOGULJAEVA,V. M., AND SEREBRYANI. S. B. 1966. Invratigntion of the primary structurp of polyhedral prokin of thr nuclear polylrcdrosis virus of silkworm. C’kr. Khim. Zh.. 32, 875-878. (In Russian.) Bozr>ov, E. A., SOGULJAEV.4. V. M., LEVITINA, T. I,., VERESCHAK, V.: AKD SERRBRYAXI, S, R. 1969. Purification of polyhedral protein of nuclear polyhedrosis virus of silkworm and the pro-
R.
WlO?+i
AND
G.
WdOndhJ
101
cess of its association in solution. Biokhimiyn, 34, 679-685. (In Russian with English summary’.) LoxGWOIWII, .J. F.. ROBERTSON, J. S., AND PAYNE:, C. c’. 1972. Thr lkfication :rnd properties of inclusion body protck of thr granulosis virus of Pieris 00/ssicfle. J. Ztl~lrrlebr. F’crthol., 19, 42-50.
I
