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Ribonucleoside triphosphatase in rabbit reticulocytes
ARCHIVES
OF
BIOCHEMISTRY
Ribonucleoside
AND
BIOPHYSICS
113, 367-370 (1966)
Triphosphatase
I. D. RAACKE,l
in Rabbit
J. FIALA,2
AND
Reticulocytes
S. i\/lATSUSHITA3
Laboratory of Comparative Biology, Kaiser Foundation Research Institute, Richmond, California, and Space Sciences Laboratory, University of California, Berkeley, California Received
August
3, 1965
Extracts of rabbit reticulocytes contain enzymes capable of specifically releasing inorganic phosphate from all four ribonucleoside triphosphates. The activities are concentrated in the ribosomal fraction, albeit to different ext,ents. They are firmly bound and cannot be removed by the usual washing procedures. The activities sediment, in a series of well-defined peaks in a sucrose density gradient. The relative proportions of the different activities, however, are not, the same in different parts of the gradient, indicating that separate enzymes are responsible for each activity. The enzymes are also activated to different extents by Mg++, Mn++, and Ca++. The ATPase differs in properties from the mitochondrial enzyme and from the membrane-bound ATPase of the Na+ and K+ transport systems. The reticulocyte triphosphatases are similar to ribosome-bound enzymes present in Escherichia coli and pea seedlings.
In earlier communications from this laboratory we have reported on specific nucleoside triphosphatases in extracts of E. coli (8) and pea seedlings (7, 9). We have now demo&rated these enzymes also in rabbit reticulocytes; and their distribution in the extracts; their behavior in the purification of the amino acid incorporating system, and some of their properties form the substance of the present report. EXPERIMENTAL Rabbit reticulocytes were obtained by a modification of the method of Borsook et al. (3), and were lysed in 0.002 M MgClz bluffer according to Allen and Schweet (1). Ribosomes, once pelleted (1X) and twice pelleted (2X), and “supernatant,” “regular,” and “purified” errzgmes were all prepared according to Hardesty et al. (4). Crude extracts and ribosomes were centrifuged in a Spinco SW 25.1 1 Present address: Department of Biology, Boston University, Boston, Massachusetts. 2 Present address: San Francisco General Hospital, San Francisco, California. 3 Present address: The Institute of Food Research, Kyoto University, Kyoto, Japan.
swinging bucket rotor for 150 minutes at 25,000 rpm on 15-307, linear sucrose density gradients (SDG), containing either 10 mM tris, pH 7.5, 10 mM KCl, and 1.5 rniV MgC12 (TKM buffer, cf. lo), or 5 mM tris, 1 mM MgClz (TM buffer). Optimal resolution of the gradients was obtained with 4-5 rng of protein per tube. At the higher concentrations necessary to provide enough material for assays, the 80s peak was only imperfectly separated from the polysome peaks. The limiting amount of protein per gradient was 30 mg, since more concentrated solutions sank in the gradient. RNA was estimated by absorption at 260 rnp, taking the optical densit,y of a O.lyA solution to be 24. Protein was determined by a micromodification of the method of Lowry et al. (5), 0.1 OD unit corresponding to 7 kg of bovine serum albumin per milliliter. On this basis, 1X and 2X ribosomes had an RNA t,o protein ratio of 0.50 + 0.08 and 0.65 + 0.09, respectively; peak tubes in the gradient had an average ratio of 0.9. Hemoglobin was estimated by absorption at 540 mr and was found to be confined to the top two tubes in the gradient, even with large loads. Ribosomes were checked for amino acid incorporating activity both before and after SDG centrifugation, and were found fully active (cj. 4). Triphosphatase activity was assayed in trip-
367
368
RAACKE,
FIALB,
AND
MATSUSHITA
of the crude extracts, whereas that of the ribosomal supernatant fraction is lower. Furthermore, both “regular” and “purified” enzymes for amino acid incorporation (4) iupernatant had an enhanced triphosphatase activit,y fraction ~-.. -compared with the supernatant fraction from which they were obtained. 19 f 10 AT1 35 * 13 The SDG sedimentation behavior of the CTP 48 zt 9 104 f 10 82 zt 20 21 + 13 triphosphatases in crude extracts could not UTP 37 z.t 5 65 * 1 1 50 xt 17 44 31 25 be studied because the amount of protein in the ribosomal region amounted to only lO15 7c of the total and did not provide enough material for assay by our usual method. In the ribosomes, however, the four activities 1.8 sedimented in a series of well-defined peaks, as shown in Fig. 1 for 1 X- ribosomes and 2 Fig. 2 for 2X- ribosomes. 1.2 i It is interest,ing to note that washing the ribosomes does not remove enzymic activity Z ii equally from all ribosomal components. This 0.8 6 is brought out more clearly in Table II, which summarizes the differences in the E relative SDG distributions of material in t’he (3 0.4 2 two types of ribosomes; t,he redistribution of RP\TA and protein among different fractions indicates t,he degree of purification in going from 1 X to 2 X . For any one triphos5 IO 15 20 25 30 phatase it is seen t,hat, after washing the activity is enhanced in some fractions and TUBE NUMBER decreased in others. However, the average 1. Distribution of ATPase (X--X), FIG. specific activity of the material in the CTPase (O-O) and protein (-) after SDG gradient was consistently higher for the centrifugation of 1 X -ribosomes. Tube 25 corre2X- than for Dhe 1 X-ribosomes, as shown in sponds to the 80s peak. t,he last column of Table II; the opposite result was obtained when whole ribosomal licate by determining the release of inorganic phosphate (8) in an incubation mixture containing pellets were assayed (Table I). 50 mM tris, pII 8.2, 5 mill KF, 5 mM MgCL, and The affinity for a given ribosomal fraction, one of the following: 2 mM ATP, 1 mM GTP, on the other hand, varies with the type of 1 mM CTP, or 1 rnhf UTP. Double-strength activity under study. The specific activity incubation mixture was added to an equal volume of ATPase, for example, is seen to be of enzyme solution containing 2-3 mg protein, or decreased in the heavy fractions of 2X0.2 ml of gradient fractions. Corrections were ribosomes, whereas t)hat of the other three made for enzyme and substrate blanks. Activities triphosphatases is increased. On the 808, are given as millimicromoles of Pi released per UTPase is also decreased, but GTPase and milligram protein in 40 minutes at 37”, rt the CTPase are enhanced. average deviation of the mean of 3-9 different preparations. The triphosphat,ases of rabbit reticulocytes require assay at 37”, since essentially RESULTS no activity can be discerned after one hour Table I shows the triphosphatase activity at 28”. The activities arc proportional to of whole crude extracts as well as that of time only during the first 40 minutes of crude and purified ribosomes and super- incubation, after which the reaction seemed natant fractions. It is seen that the specific to slow down or even stop abruptly. activity of t’he ribosomes is higher than that Enzyme activity is dependent on the TABLE
TRIPHOSPH.IT.~SE RETICCLOCYTE
~~‘YE&2‘2
I
ACTIVITIES FRACTIONS
IK
= ;2
TRIPHOSPHATASES
1
5
:w
IN RETICULOCYTES
IO TUBE
15 NUMBER
20
25
FIG. 2. Distribution of ATPase (X--X), CTPase (O-O), GTPase (O-O), UTPase (A--A), and protein (-) after SDG centrifugation of 2X-ribosomes. Tube 21 corresponds to the 80s peak. TABLE RELATIVE
DISTRIBUTIONS OF RNA, PROTEIN, RETICULOCYTE RIBOSOMES
II
AND TRIPHOSPHATASES IN AFTER SDG CENTRIFUGATION
Avg. % totalC
Activity/mg
Fractions”
Type of analysis
CRUDE
AND
PURIFIED
protein
Fractions
Rib.
I
II
III
IV
I
II
III
IV
Total
Proteinb
1X 2x
7 13
26 50
10 16
60 21
RNA
1x 2x
12 12
48 59
16 19
24 10
0.72 0.70
0.90 0.87
0.74 0.89
0.19 0.39
0.46 0.73
ATPase
1x 2x
22 30
46 49
7 6
24 16
435 320
261 149
159 84
73 237
163 180
GTPase
1X 2x
26 30
30 45
8 12
36 13
734 620
179 205
85 122
68 81
121 191
CTPase
1x 2X
20 27
34 45
12 12
35 17
461 578
184 240
154 193
74 208
132 250
UTPase
1x 2x
17 34
33 47
13 8
37 11
325 591
135 182
94 71
48 57
82 162
5 The fractions represent the following: I, heavy material, approximately the bottom 10 ml; II, main polysome peak, next 12 ml; III, 80s ribosomes, 3 ml; and IV, the top 5 ml containing small ribosomes and supernatant fraction. Analyses were performed for each individual tube and averaged over each fraction. b These runs were made in TM buffer. In TKM buffer there is slightly more RNA and considerably more protein in fractions I and II, and correspondingly less in fractions III and IV. c Total refers to amount of material or activities recovered from the gradient in each experiment. The recovery for both RNA and protein averaged 95% f 10 for 9 runs. Recovery of activities for IXribosomes was close to complete, but 2X-ribosomes suffered a a-fold activation after SDG.
370
RAACKE, TABLE
FIALA,
III
EFFECTS OF DIFFERENT
METALS ON TRIPHOSPHATASE ACTIVITIES IN CRUDE EXTR.ICTH OF RETICCLOCYTES Activator Mg++ Mn++ Ca++
Substlate ATP
GTP
CTP
UTP
47 24 40
50 61 32
40 49 30
41 45 45
presence of a divalent cation. Table III shows the differential activations of the four triphosphatases by Mg++, h!tn++, and Ca++. DISCUSSION
Of the four ribonucleoside triphosphatase activities only GTPase has been reported to be present in rabbit reticulocytes. Part of the latter has been implicated by Arlinghaus et al. (2) in the enzymic binding of aminoacyl-sRNA to ribosomes, preparatory to peptide bond formation, but the larger part’ of the activity could not be associated with any specific function. Similarly, no function can yet be assigned to the ribosome-bound CTPase, UTPase, and ATPase activities. An ATPase activity associated with Nat and I<+ transport has been described in human erythrocytes (B), but, it is membranebound and differs in other properties from t’he cytoplasmic ATPase here described. Triphosphatases similar to those in reticulocytes are present in bact,erial (8) and plant cells (7). These enzymes therefore seem to be of general occurrence. The bulk of the triphosphatase activity in all t’hree systems is bound to ribonucleoprotein, but’ in E. coli the association is labile and easily broken by washing, whereas in both reticulocytes and pea seedlings the enzymes are so tight’ly bound that washing increases rat’her than decreases the specific act,iv&y of t’he ribosomes. Furthermore, t’he
AND
MATSUSHITA
enzymes in all t’hree systems sediment in sharp peaks in the sucrose gradient. In reticulocyt’es, as in other systems, there seem to be four different enzymes, as indicat,ed by the differential affinity of different activities for different ribosomal components (Tables I and II) as well as by the differential effect, of metal ions (Table III). However, the reticulocyte enzymes are about fifty t,imes less active on a prot’ein basis than those in E:. coli, and three orders of magnitude less than t’hose of pea seedlings. Furthermore, the animal enzymes require a higher temperature for activit,y than those from plant,s and bacteria. ACKNOWLEDGMENTS This work was supported hv grants GM-07924-02 from the National Institutes of Health, GB-2076 from the National Science Foundation, and NSG 479 from the National Aeronautics and Space Agency. REFERENCES 1. ALLEN, E. H., AND SCHXEET, R. S., J. Biol. Chem. 237, 760 (1962). 2. ARLINGHAUS, It., AND SCH.IEFFER, J., SCHWEET, K. S., Proc. Xatl. Acad. Sci. U.S. 61, 1291 (1964j. 3. BORSOOK, H., DEGY, C. L., &~GEN-SMIT, A. J., KEIGHLEY, G., ~SD Lowu, P. H., J. Biol. Chem. 196, 669 (1952). 4. HSRDESTY, B., MILLER, R.,.&ND SCHWEET,~., Proc. Xatl. Acud. Sci. r.8. 50, 924 (1963). 5. LOIYRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951j. 0. POST, R. I,., MERRITT, C. R., KINSOLVING, C. R., AND ALBRIGHT, C. D., J. Biol. Chem. 236, 1796 (1960). 7. RAACKE, I. I)., Federation Proc. 22, 348 (1963). 8. RA4~c~~, I. D., END FI.IL.I, J., Proc. Satl. Acad. Sci. C.S. 61, 323 (1964). 9. R.~ACKE, I. D., 2~zs~)&~ITSUSHIT.I, S., Federation Proc. 23, 532 (1964). LO. WARNER, J. R., KNOPF, P. M., AND RICH, A., Proc. 12’atl. dcad. Sci. I’.S. 49, 122 (1963).
OF
BIOCHEMISTRY
Ribonucleoside
AND
BIOPHYSICS
113, 367-370 (1966)
Triphosphatase
I. D. RAACKE,l
in Rabbit
J. FIALA,2
AND
Reticulocytes
S. i\/lATSUSHITA3
Laboratory of Comparative Biology, Kaiser Foundation Research Institute, Richmond, California, and Space Sciences Laboratory, University of California, Berkeley, California Received
August
3, 1965
Extracts of rabbit reticulocytes contain enzymes capable of specifically releasing inorganic phosphate from all four ribonucleoside triphosphates. The activities are concentrated in the ribosomal fraction, albeit to different ext,ents. They are firmly bound and cannot be removed by the usual washing procedures. The activities sediment, in a series of well-defined peaks in a sucrose density gradient. The relative proportions of the different activities, however, are not, the same in different parts of the gradient, indicating that separate enzymes are responsible for each activity. The enzymes are also activated to different extents by Mg++, Mn++, and Ca++. The ATPase differs in properties from the mitochondrial enzyme and from the membrane-bound ATPase of the Na+ and K+ transport systems. The reticulocyte triphosphatases are similar to ribosome-bound enzymes present in Escherichia coli and pea seedlings.
In earlier communications from this laboratory we have reported on specific nucleoside triphosphatases in extracts of E. coli (8) and pea seedlings (7, 9). We have now demo&rated these enzymes also in rabbit reticulocytes; and their distribution in the extracts; their behavior in the purification of the amino acid incorporating system, and some of their properties form the substance of the present report. EXPERIMENTAL Rabbit reticulocytes were obtained by a modification of the method of Borsook et al. (3), and were lysed in 0.002 M MgClz bluffer according to Allen and Schweet (1). Ribosomes, once pelleted (1X) and twice pelleted (2X), and “supernatant,” “regular,” and “purified” errzgmes were all prepared according to Hardesty et al. (4). Crude extracts and ribosomes were centrifuged in a Spinco SW 25.1 1 Present address: Department of Biology, Boston University, Boston, Massachusetts. 2 Present address: San Francisco General Hospital, San Francisco, California. 3 Present address: The Institute of Food Research, Kyoto University, Kyoto, Japan.
swinging bucket rotor for 150 minutes at 25,000 rpm on 15-307, linear sucrose density gradients (SDG), containing either 10 mM tris, pH 7.5, 10 mM KCl, and 1.5 rniV MgC12 (TKM buffer, cf. lo), or 5 mM tris, 1 mM MgClz (TM buffer). Optimal resolution of the gradients was obtained with 4-5 rng of protein per tube. At the higher concentrations necessary to provide enough material for assays, the 80s peak was only imperfectly separated from the polysome peaks. The limiting amount of protein per gradient was 30 mg, since more concentrated solutions sank in the gradient. RNA was estimated by absorption at 260 rnp, taking the optical densit,y of a O.lyA solution to be 24. Protein was determined by a micromodification of the method of Lowry et al. (5), 0.1 OD unit corresponding to 7 kg of bovine serum albumin per milliliter. On this basis, 1X and 2X ribosomes had an RNA t,o protein ratio of 0.50 + 0.08 and 0.65 + 0.09, respectively; peak tubes in the gradient had an average ratio of 0.9. Hemoglobin was estimated by absorption at 540 mr and was found to be confined to the top two tubes in the gradient, even with large loads. Ribosomes were checked for amino acid incorporating activity both before and after SDG centrifugation, and were found fully active (cj. 4). Triphosphatase activity was assayed in trip-
367
368
RAACKE,
FIALB,
AND
MATSUSHITA
of the crude extracts, whereas that of the ribosomal supernatant fraction is lower. Furthermore, both “regular” and “purified” enzymes for amino acid incorporation (4) iupernatant had an enhanced triphosphatase activit,y fraction ~-.. -compared with the supernatant fraction from which they were obtained. 19 f 10 AT1 35 * 13 The SDG sedimentation behavior of the CTP 48 zt 9 104 f 10 82 zt 20 21 + 13 triphosphatases in crude extracts could not UTP 37 z.t 5 65 * 1 1 50 xt 17 44 31 25 be studied because the amount of protein in the ribosomal region amounted to only lO15 7c of the total and did not provide enough material for assay by our usual method. In the ribosomes, however, the four activities 1.8 sedimented in a series of well-defined peaks, as shown in Fig. 1 for 1 X- ribosomes and 2 Fig. 2 for 2X- ribosomes. 1.2 i It is interest,ing to note that washing the ribosomes does not remove enzymic activity Z ii equally from all ribosomal components. This 0.8 6 is brought out more clearly in Table II, which summarizes the differences in the E relative SDG distributions of material in t’he (3 0.4 2 two types of ribosomes; t,he redistribution of RP\TA and protein among different fractions indicates t,he degree of purification in going from 1 X to 2 X . For any one triphos5 IO 15 20 25 30 phatase it is seen t,hat, after washing the activity is enhanced in some fractions and TUBE NUMBER decreased in others. However, the average 1. Distribution of ATPase (X--X), FIG. specific activity of the material in the CTPase (O-O) and protein (-) after SDG gradient was consistently higher for the centrifugation of 1 X -ribosomes. Tube 25 corre2X- than for Dhe 1 X-ribosomes, as shown in sponds to the 80s peak. t,he last column of Table II; the opposite result was obtained when whole ribosomal licate by determining the release of inorganic phosphate (8) in an incubation mixture containing pellets were assayed (Table I). 50 mM tris, pII 8.2, 5 mill KF, 5 mM MgCL, and The affinity for a given ribosomal fraction, one of the following: 2 mM ATP, 1 mM GTP, on the other hand, varies with the type of 1 mM CTP, or 1 rnhf UTP. Double-strength activity under study. The specific activity incubation mixture was added to an equal volume of ATPase, for example, is seen to be of enzyme solution containing 2-3 mg protein, or decreased in the heavy fractions of 2X0.2 ml of gradient fractions. Corrections were ribosomes, whereas t)hat of the other three made for enzyme and substrate blanks. Activities triphosphatases is increased. On the 808, are given as millimicromoles of Pi released per UTPase is also decreased, but GTPase and milligram protein in 40 minutes at 37”, rt the CTPase are enhanced. average deviation of the mean of 3-9 different preparations. The triphosphat,ases of rabbit reticulocytes require assay at 37”, since essentially RESULTS no activity can be discerned after one hour Table I shows the triphosphatase activity at 28”. The activities arc proportional to of whole crude extracts as well as that of time only during the first 40 minutes of crude and purified ribosomes and super- incubation, after which the reaction seemed natant fractions. It is seen that the specific to slow down or even stop abruptly. activity of t’he ribosomes is higher than that Enzyme activity is dependent on the TABLE
TRIPHOSPH.IT.~SE RETICCLOCYTE
~~‘YE&2‘2
I
ACTIVITIES FRACTIONS
IK
= ;2
TRIPHOSPHATASES
1
5
:w
IN RETICULOCYTES
IO TUBE
15 NUMBER
20
25
FIG. 2. Distribution of ATPase (X--X), CTPase (O-O), GTPase (O-O), UTPase (A--A), and protein (-) after SDG centrifugation of 2X-ribosomes. Tube 21 corresponds to the 80s peak. TABLE RELATIVE
DISTRIBUTIONS OF RNA, PROTEIN, RETICULOCYTE RIBOSOMES
II
AND TRIPHOSPHATASES IN AFTER SDG CENTRIFUGATION
Avg. % totalC
Activity/mg
Fractions”
Type of analysis
CRUDE
AND
PURIFIED
protein
Fractions
Rib.
I
II
III
IV
I
II
III
IV
Total
Proteinb
1X 2x
7 13
26 50
10 16
60 21
RNA
1x 2x
12 12
48 59
16 19
24 10
0.72 0.70
0.90 0.87
0.74 0.89
0.19 0.39
0.46 0.73
ATPase
1x 2x
22 30
46 49
7 6
24 16
435 320
261 149
159 84
73 237
163 180
GTPase
1X 2x
26 30
30 45
8 12
36 13
734 620
179 205
85 122
68 81
121 191
CTPase
1x 2X
20 27
34 45
12 12
35 17
461 578
184 240
154 193
74 208
132 250
UTPase
1x 2x
17 34
33 47
13 8
37 11
325 591
135 182
94 71
48 57
82 162
5 The fractions represent the following: I, heavy material, approximately the bottom 10 ml; II, main polysome peak, next 12 ml; III, 80s ribosomes, 3 ml; and IV, the top 5 ml containing small ribosomes and supernatant fraction. Analyses were performed for each individual tube and averaged over each fraction. b These runs were made in TM buffer. In TKM buffer there is slightly more RNA and considerably more protein in fractions I and II, and correspondingly less in fractions III and IV. c Total refers to amount of material or activities recovered from the gradient in each experiment. The recovery for both RNA and protein averaged 95% f 10 for 9 runs. Recovery of activities for IXribosomes was close to complete, but 2X-ribosomes suffered a a-fold activation after SDG.
370
RAACKE, TABLE
FIALA,
III
EFFECTS OF DIFFERENT
METALS ON TRIPHOSPHATASE ACTIVITIES IN CRUDE EXTR.ICTH OF RETICCLOCYTES Activator Mg++ Mn++ Ca++
Substlate ATP
GTP
CTP
UTP
47 24 40
50 61 32
40 49 30
41 45 45
presence of a divalent cation. Table III shows the differential activations of the four triphosphatases by Mg++, h!tn++, and Ca++. DISCUSSION
Of the four ribonucleoside triphosphatase activities only GTPase has been reported to be present in rabbit reticulocytes. Part of the latter has been implicated by Arlinghaus et al. (2) in the enzymic binding of aminoacyl-sRNA to ribosomes, preparatory to peptide bond formation, but the larger part’ of the activity could not be associated with any specific function. Similarly, no function can yet be assigned to the ribosome-bound CTPase, UTPase, and ATPase activities. An ATPase activity associated with Nat and I<+ transport has been described in human erythrocytes (B), but, it is membranebound and differs in other properties from t’he cytoplasmic ATPase here described. Triphosphatases similar to those in reticulocytes are present in bact,erial (8) and plant cells (7). These enzymes therefore seem to be of general occurrence. The bulk of the triphosphatase activity in all t’hree systems is bound to ribonucleoprotein, but’ in E. coli the association is labile and easily broken by washing, whereas in both reticulocytes and pea seedlings the enzymes are so tight’ly bound that washing increases rat’her than decreases the specific act,iv&y of t’he ribosomes. Furthermore, t’he
AND
MATSUSHITA
enzymes in all t’hree systems sediment in sharp peaks in the sucrose gradient. In reticulocyt’es, as in other systems, there seem to be four different enzymes, as indicat,ed by the differential affinity of different activities for different ribosomal components (Tables I and II) as well as by the differential effect, of metal ions (Table III). However, the reticulocyte enzymes are about fifty t,imes less active on a prot’ein basis than those in E:. coli, and three orders of magnitude less than t’hose of pea seedlings. Furthermore, the animal enzymes require a higher temperature for activit,y than those from plant,s and bacteria. ACKNOWLEDGMENTS This work was supported hv grants GM-07924-02 from the National Institutes of Health, GB-2076 from the National Science Foundation, and NSG 479 from the National Aeronautics and Space Agency. REFERENCES 1. ALLEN, E. H., AND SCHXEET, R. S., J. Biol. Chem. 237, 760 (1962). 2. ARLINGHAUS, It., AND SCH.IEFFER, J., SCHWEET, K. S., Proc. Xatl. Acad. Sci. U.S. 61, 1291 (1964j. 3. BORSOOK, H., DEGY, C. L., &~GEN-SMIT, A. J., KEIGHLEY, G., ~SD Lowu, P. H., J. Biol. Chem. 196, 669 (1952). 4. HSRDESTY, B., MILLER, R.,.&ND SCHWEET,~., Proc. Xatl. Acud. Sci. r.8. 50, 924 (1963). 5. LOIYRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951j. 0. POST, R. I,., MERRITT, C. R., KINSOLVING, C. R., AND ALBRIGHT, C. D., J. Biol. Chem. 236, 1796 (1960). 7. RAACKE, I. I)., Federation Proc. 22, 348 (1963). 8. RA4~c~~, I. D., END FI.IL.I, J., Proc. Satl. Acad. Sci. C.S. 61, 323 (1964). 9. R.~ACKE, I. D., 2~zs~)&~ITSUSHIT.I, S., Federation Proc. 23, 532 (1964). LO. WARNER, J. R., KNOPF, P. M., AND RICH, A., Proc. 12’atl. dcad. Sci. I’.S. 49, 122 (1963).