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Separation and determination of cross-linking amino acids of elastin by high-voltage paper electrophoresis
ANALYTICAL
45, 422427
BIOCHEMISTRY
Separation
and Acids
Determination of Elastin Paper
E. MOCZAR, Laboratoire
(1972)
of Cross-Linking by High-Voltage
Electrophoresis
B. ROBERT,
de Biochimie
Amino
du Tissu
Y6 PARIS
l.Je,
AND
L. ROBERT
Conjonctif,
6ter
rue
~Albia,
France
Received May 3, 1971
Purification of elastin can be accomplished by hydrolytic procedures using acids or alkali, enzymes, or autoclaving (l-3). The resulting preparations can be standardized and characterized only by their amino acid composition (1,4,5) and mainly by their desmosine and isodesmosine contents (6-9). These cross-linking amino acids are the only specific earmarks of elastin. Some other cross-linking amino acids are also present, as lysinonorleucine (10) and an intermediary product of desmosine which can be converted to merodesmosine (11). However the former is present in collagen also (12,13), and is probably identical with the aldol condensation product of two allysines, yielding on reduction a derivative which was isolated and characterized (14). These considerations render important the specific determination of these cross-linking amino acids of elastin. The methods proposed for desmosine determination all use the amino acid analyzer (6-9). We propose here a simpler and faster procedure which enabled us to carry out serial determinations of cross-linking amino acids in several elastin preparations purified by different methods (3). This procedure is based on the separation of the cross-linking amino acids from the neutral, acid, and basic amino acids by high-voltage electrophoresis. The ninhydrincoloured strips are then quantitated by densitometric recording. The same method can be used for the isolation of small quantities of cross-linking amino acids by preparative electrophoresis followed by elution of the spots from the paper. This method enabled us to carry out the characterization of some of the isolated substances by thin-layer chromatography and by mass spectrometry (15). MATERIALS
AND
METHODS
The preparation and purification of elastin samples were carried out as described earlier (3,15). Purified elastin obtained from beef ligamenturn 422 @ 1972 by Academic
Press, Inc.
CROSS-LINKING
AMINO
ACIDS
OF
423
CLASTIN
nuchae and from pig, calf, horse, or human aorta was washed in acetone, dried at 37” and in the desiccator over PZ05. Its water and ash content were determined and the amino acid contents were corrected accordingly. Samples of 5 to 10 mg were hydrolyzed in 6N HCl for 48 hr at 110” in constant-boiling HCl in vacuum-sealed tubes; the acid hydrolyzates were dried in vacuum over KOH and dissolved in water to a final concentration of 1%. Whatman 3 or 3MM paper was used in a Gilson Electrophorator model D. From the solutions prepared as described above 20 ~1 was deposited on a 8-10 mm length of the starting line. The paper was sprayed by the buffer, pyridineJacetic acid/water (1:10:89 v/v/v), pH 3.8, and a voltage of 5000V was applied for 1.5 hr. The air-dried paper was dipped in the ninhydrin cadmium reagent (16) and colour was developed in the dark for 24 hr at room temperature. The intensities of the revealed spots were estimated by photodensitometry on a Photovolt Densicord apparatus. Peak areas were calculated by multiplying the height of the peaks by their width at midheight or by planimetry. Another method of quantitative estimation consisted in cutting out the ninhydrin-stained spots and eluting the pigment in methanol. The optical density of the methanol extract was then read at 500 rnp on a Beckman spectrophotometer. This second method is more sensitive and was used for the quantitative evaluation of the minor spots. In preparative runs only a marginal band was st’ained, the rest of the paper being cut out accordingly and eluted by descending chromatography using 0.05 N HCl as eluant. The collected eluate was evaporated under vacuum. Thin-layer chromatography was carried out on microcrystalline cellulose (Selecta-144, Schleicher & Schiill) using the following solvents: (1) Pyridine/ethyl (2) n-butanol/acetic The thin-layer procedure.
acetate/acetic acid/water
chromatograms
acid/water (5:5:1:3 (12:3:5 v/v).
were also stained
v/v).
by the same ninhydrin
RESULTS Figure 1 shows a typical high-voltage run of several elastin hydrolyzates. Eight different spots could be detected in the region corresponding to the weakly basic amino acids. Their electrophoretic mobilities related to lysine, taken as one (glucose being used to correct for the electroosmotic flow), are given in Table 1, as well as the chemical identity of some spots determined as described below together with
424
MOCZAR,
ROBERT,
AND
ROBERT
FIG. 1. High-voltage electrophoresis of several elastin hydrolyzates. Asp = aspartic acid. The numbers correspond to the weakly basic amino acids (see Table 1). 9 being lysine and histidine. Samples (refers to figures shown on the starting line) : (1) elastin from human aorta purified by NaOH extraction, (2) elastin from beef ligamentum nuchae purified by NaOH extraction, (3) elastin from human aorta purified by extraction with 70% trichloroacetic acid (3).
their relative concentration (related to aspartic acid) in two NaOHpurified elastin preparations from beef ligamenturn nuchae and from human aorta. Figure 2 is the densitometric recording of the electropherogram shown in Fig. 1. The calculated quantities for the ninhydrin-positive substances
Separation and Identification Electrophoresis at pH
TABLE 1 of Cross-Linking Amino 3.8 of Two Elastin Samples Extraction (3,14)
Acids by High-Voltage Purified by NaOH
Rel. amt. “cross-linking” acids in two NaOH purified samples
Spot NO.Q 1 2 3 4 5 6 7 8 9
Rel. migration rate* 0.39 0.43 0.47 0.53 0.55 0.61 0.66 0.71 1.00
Ratio taken Nature
Desmosine
of substance
and isodesmosine
Lysinonorleucine Lysinoalanine Lysine (hiat. p
A 0.12 0.12 0.75 0.09 0.06 0.08 0.13 0.31 0.72
Sample A, from human aorta (rmoles asp./100 mg, 16.3). mentum nuchae (@moles asp./100 mg, 6.5). a See Fig. 1. * Related to lysine and corrected for electro-osmotic flow. c Lysine + histidine migrating together.
to asp. as unity B 0.20 0.20 1.40 0.10 0.22 traces 0.37 0.17 1.2 Sample
amino elastin
~mole/fimole asp. A
B
0.73
1.38
0.12
0.36
B, from
beef liga-
425
GO 30 20 10 -
O/
I
3
2. Densitometric recording of high voltage electrophoresis of NaOH-purified clastin sample. The numbers correspond to the spots as shown on Fig. 1. Abscissa: distance traveled by the spot. Ordinate: absorbance of ninhydrin-colored spots. FIG.
present in the eight peaks are given in Table 1. The desmosine plus isodesmosine contents calculated from these recordings are in good agreement with determinations carried out on t,he amino acid analyzer. The sample of aorta elastin shown in Table 1 gave on the amino acid. analyzer 2.6 residues per 1000 of desmosineplus isodesmosine (expressed in lysine equivalents) and 3.3 residues per 1000 of aspartic acid (5). The ratio of desmosine+ isodesmosine to aspartic acid is 0.78, which compares favorably with the ratio of 0.73 found by the present method. The same. ratio for ligamenturn nuchae elastin was found to be 1.28 on the amino acid analyzer and 1.38 by the present method. Similar determinations carried out on several elastin samples from aorta and ligamenturn nuchae purified by different methods gave very similar patterns to those shown in Fig. 1 and made possible a detailed study of the distribution of cross-linking amino acids in these elastin samples (15). Identification of the demosines and lysinonorleucine ,in the separated spots. All the eight separated spots eluted from a preparative electrophoretic run were concentrated under vacuum and rechromatographed on thin-layer microcrystalline cellulose in two solvents. Only one spot could be detected in each of the eight substances. Amino acids present in spot 3 on high-voltage runs could be identified as a mixture of desmosine and isodesmosine and in spot 7 as lysinonorleucine by comparing their electrophoretic and chromatographic mobilities to that of authentic samples. The other peaks have not yet been identified with certainty. Quczntitative estimation of desmosines. The colour intensities (peak areas) obtained with the ninhydrin-cadmium reaction of desmosine, isodesmosine, and lysinonorleucine are in a linear relationship to the
426
MOCZAR,
ROBERT,
AND
ROBERT
weight of the substances deposited up to 3 pg. The colour yield (expressed as lysine equivalents) of desmosine and isodesmosine with ninhydrin is 0.85-0.90, of lysinonorleucine 0.7, related to that of aspartic acid. Aspartic acid is convenient for comparison because it is the best separated amino acid on the electrophoretic runs under these working conditions. The same ratio is about 0.7 and 0.5, respectively, related to leucine colour. The standard error of the mean of 3 repeated determinations is of the order of 6-7s for the main peaks (aspartic acid, desmosine, isodesmosine, lysinonorleucine) and about 10% for the minor spots. DISCUSSION The reported method is simple and reliable and gives reproducible results on small samples. It can be used for the characterization of elastin samples as well as for the isolation and characterization of some of the less well known cross-linking amino acids (15). Determination of the specific activity of radioactive spots could also be carried out on elastin preparations of aortas incubated with W-lysine (17). One of the drawbacks of the method, as compared to the amino acid analyzer, is the lack of separation of desmosine and isodesmosine. Paper electrophoresis at 300 V under standard conditions appears to be sufficient to separate the desmosines from most of the other amino acids, as was shown by Blass (18). High voltage is, however, necessary for the separation of the other weakly basic amino acids participating in the cross-linking of elastin and collagen. A similar method enabling the separation and quantitative determination of hydroxylysine and its glycosides in collagen hydrolyzate was recently reported from this laboratory (19). These two micro methods together were helpful in the quantitative analytical and metabolic studies on collagen and elastin (15,17). SUMMARY A method is described for the separation and quantitative determination of the weakly basic (cross-linking) amino acids of elastin. A 6 N HCl hydrolyzate is submitted to high-voltage electrophoresis at pH 3.8. At least eight spots can be identified in the weakly basic region and quantitated by the ninhydrin-photodensitometric method. Some of these are spot 3 for desmosine + isodesmosine, and 7 for lysinonorleutine. Quantitative data given for two typical elastin preparations are in agreement with direct amino acid analysis. Preparative electrophoresis can also be carried out and used for the isolation of small quantities of the unknown spots separated by this procedure.
CROSS-LIA-KIiYG
AMINO
ACIDS
OF CLASTIN
427
ACKNOWLEDGMENTS Supported by CNRS and We would like to express Institute, Langford, England) and to Dr. J. Blass (In&it&
DGRST. our gratitude to Dr. S. M. Partridge (Meat Research for the gift of a sample of desmosine and isodesmosine Pasteur, Garches) for a sample of lysinonorleucine. REFERENCES
M., Advan. Protein Chem. 17, 227 (1962). “Structure and Function of Elastin and Collagen.” AkadCmiai Kiad6, Budapest, 1966. ROBERT, L., ROBERT, B., AND ROBERT, A. M., Exp. Gerontol. 5, 339 (1970). MANDL, I., KELLER, S., AND LEVI. M., in “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs. ed.), Vol. 1, p. 657. Academic Press, New York, 1970. COMTE, P., AND ROBERT, L., Bull. Sot. Chim. Biol. 50, 1349 (1968). GOTTE, L., STERN, P., ELSDEN, D. T.. AND PARTRIDGE, S. M., Biochem. J. 87, 344 (1963). CORBIN, K. W., Anal. Biochem. 32, 118 (1969). LEDVINA, M., AND BARTOS, F., J. Chromatogr. 31, 56 (1967). ANWAR, R. A., Can. J. Biochem. 44, 725 (1966). FRANZBLAU, C., FARIS, B., AND PAPOIOANNON, R., Biochemistry 8, 2833 (1969). STARCHER, B. C., PARTRIDGE, S. M., AKD ELSDEN, D. F., Biochemistry 6, 2425 (1967). BAILEY, A. J., PEACH, C. M., AND FOWLER, L. J., Biochemistry 117, 819 (1970). KANG, A. H., FARIS, B., AND FRANZBLAU. C., Biochem. Biophys. Res. Commun. 36, 345 (1969). LENT, R. W., SMITH, B., SALCEDO. L. L., FARIS, B., AND FRANZBLAU, C., Biochemistry 8, 2837 (1969). MOCZAR, E., ROBERT, B., DEROUETTE, J. C., AND ROBERT, L., in preparation. HEITMANN, J., BARROLIER, J., AND WATZKE, E., 2. Physiol. Chem. 309, 219 (1957). JUNQUA, S., ROBERT, A. M., AND ROBERT, L., in preparation. BLASS, J., C. R. Acad. Sci. Ser. C. 271, 94 (1970). MOCZAR, E., AND MOCZAR, M., J. Chromatogr. 51, 277 (1970).
1. PARTRIDGE, S. 2. BANGA, I., in
3. 4.
5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19.
45, 422427
BIOCHEMISTRY
Separation
and Acids
Determination of Elastin Paper
E. MOCZAR, Laboratoire
(1972)
of Cross-Linking by High-Voltage
Electrophoresis
B. ROBERT,
de Biochimie
Amino
du Tissu
Y6 PARIS
l.Je,
AND
L. ROBERT
Conjonctif,
6ter
rue
~Albia,
France
Received May 3, 1971
Purification of elastin can be accomplished by hydrolytic procedures using acids or alkali, enzymes, or autoclaving (l-3). The resulting preparations can be standardized and characterized only by their amino acid composition (1,4,5) and mainly by their desmosine and isodesmosine contents (6-9). These cross-linking amino acids are the only specific earmarks of elastin. Some other cross-linking amino acids are also present, as lysinonorleucine (10) and an intermediary product of desmosine which can be converted to merodesmosine (11). However the former is present in collagen also (12,13), and is probably identical with the aldol condensation product of two allysines, yielding on reduction a derivative which was isolated and characterized (14). These considerations render important the specific determination of these cross-linking amino acids of elastin. The methods proposed for desmosine determination all use the amino acid analyzer (6-9). We propose here a simpler and faster procedure which enabled us to carry out serial determinations of cross-linking amino acids in several elastin preparations purified by different methods (3). This procedure is based on the separation of the cross-linking amino acids from the neutral, acid, and basic amino acids by high-voltage electrophoresis. The ninhydrincoloured strips are then quantitated by densitometric recording. The same method can be used for the isolation of small quantities of cross-linking amino acids by preparative electrophoresis followed by elution of the spots from the paper. This method enabled us to carry out the characterization of some of the isolated substances by thin-layer chromatography and by mass spectrometry (15). MATERIALS
AND
METHODS
The preparation and purification of elastin samples were carried out as described earlier (3,15). Purified elastin obtained from beef ligamenturn 422 @ 1972 by Academic
Press, Inc.
CROSS-LINKING
AMINO
ACIDS
OF
423
CLASTIN
nuchae and from pig, calf, horse, or human aorta was washed in acetone, dried at 37” and in the desiccator over PZ05. Its water and ash content were determined and the amino acid contents were corrected accordingly. Samples of 5 to 10 mg were hydrolyzed in 6N HCl for 48 hr at 110” in constant-boiling HCl in vacuum-sealed tubes; the acid hydrolyzates were dried in vacuum over KOH and dissolved in water to a final concentration of 1%. Whatman 3 or 3MM paper was used in a Gilson Electrophorator model D. From the solutions prepared as described above 20 ~1 was deposited on a 8-10 mm length of the starting line. The paper was sprayed by the buffer, pyridineJacetic acid/water (1:10:89 v/v/v), pH 3.8, and a voltage of 5000V was applied for 1.5 hr. The air-dried paper was dipped in the ninhydrin cadmium reagent (16) and colour was developed in the dark for 24 hr at room temperature. The intensities of the revealed spots were estimated by photodensitometry on a Photovolt Densicord apparatus. Peak areas were calculated by multiplying the height of the peaks by their width at midheight or by planimetry. Another method of quantitative estimation consisted in cutting out the ninhydrin-stained spots and eluting the pigment in methanol. The optical density of the methanol extract was then read at 500 rnp on a Beckman spectrophotometer. This second method is more sensitive and was used for the quantitative evaluation of the minor spots. In preparative runs only a marginal band was st’ained, the rest of the paper being cut out accordingly and eluted by descending chromatography using 0.05 N HCl as eluant. The collected eluate was evaporated under vacuum. Thin-layer chromatography was carried out on microcrystalline cellulose (Selecta-144, Schleicher & Schiill) using the following solvents: (1) Pyridine/ethyl (2) n-butanol/acetic The thin-layer procedure.
acetate/acetic acid/water
chromatograms
acid/water (5:5:1:3 (12:3:5 v/v).
were also stained
v/v).
by the same ninhydrin
RESULTS Figure 1 shows a typical high-voltage run of several elastin hydrolyzates. Eight different spots could be detected in the region corresponding to the weakly basic amino acids. Their electrophoretic mobilities related to lysine, taken as one (glucose being used to correct for the electroosmotic flow), are given in Table 1, as well as the chemical identity of some spots determined as described below together with
424
MOCZAR,
ROBERT,
AND
ROBERT
FIG. 1. High-voltage electrophoresis of several elastin hydrolyzates. Asp = aspartic acid. The numbers correspond to the weakly basic amino acids (see Table 1). 9 being lysine and histidine. Samples (refers to figures shown on the starting line) : (1) elastin from human aorta purified by NaOH extraction, (2) elastin from beef ligamentum nuchae purified by NaOH extraction, (3) elastin from human aorta purified by extraction with 70% trichloroacetic acid (3).
their relative concentration (related to aspartic acid) in two NaOHpurified elastin preparations from beef ligamenturn nuchae and from human aorta. Figure 2 is the densitometric recording of the electropherogram shown in Fig. 1. The calculated quantities for the ninhydrin-positive substances
Separation and Identification Electrophoresis at pH
TABLE 1 of Cross-Linking Amino 3.8 of Two Elastin Samples Extraction (3,14)
Acids by High-Voltage Purified by NaOH
Rel. amt. “cross-linking” acids in two NaOH purified samples
Spot NO.Q 1 2 3 4 5 6 7 8 9
Rel. migration rate* 0.39 0.43 0.47 0.53 0.55 0.61 0.66 0.71 1.00
Ratio taken Nature
Desmosine
of substance
and isodesmosine
Lysinonorleucine Lysinoalanine Lysine (hiat. p
A 0.12 0.12 0.75 0.09 0.06 0.08 0.13 0.31 0.72
Sample A, from human aorta (rmoles asp./100 mg, 16.3). mentum nuchae (@moles asp./100 mg, 6.5). a See Fig. 1. * Related to lysine and corrected for electro-osmotic flow. c Lysine + histidine migrating together.
to asp. as unity B 0.20 0.20 1.40 0.10 0.22 traces 0.37 0.17 1.2 Sample
amino elastin
~mole/fimole asp. A
B
0.73
1.38
0.12
0.36
B, from
beef liga-
425
GO 30 20 10 -
O/
I
3
2. Densitometric recording of high voltage electrophoresis of NaOH-purified clastin sample. The numbers correspond to the spots as shown on Fig. 1. Abscissa: distance traveled by the spot. Ordinate: absorbance of ninhydrin-colored spots. FIG.
present in the eight peaks are given in Table 1. The desmosine plus isodesmosine contents calculated from these recordings are in good agreement with determinations carried out on t,he amino acid analyzer. The sample of aorta elastin shown in Table 1 gave on the amino acid. analyzer 2.6 residues per 1000 of desmosineplus isodesmosine (expressed in lysine equivalents) and 3.3 residues per 1000 of aspartic acid (5). The ratio of desmosine+ isodesmosine to aspartic acid is 0.78, which compares favorably with the ratio of 0.73 found by the present method. The same. ratio for ligamenturn nuchae elastin was found to be 1.28 on the amino acid analyzer and 1.38 by the present method. Similar determinations carried out on several elastin samples from aorta and ligamenturn nuchae purified by different methods gave very similar patterns to those shown in Fig. 1 and made possible a detailed study of the distribution of cross-linking amino acids in these elastin samples (15). Identification of the demosines and lysinonorleucine ,in the separated spots. All the eight separated spots eluted from a preparative electrophoretic run were concentrated under vacuum and rechromatographed on thin-layer microcrystalline cellulose in two solvents. Only one spot could be detected in each of the eight substances. Amino acids present in spot 3 on high-voltage runs could be identified as a mixture of desmosine and isodesmosine and in spot 7 as lysinonorleucine by comparing their electrophoretic and chromatographic mobilities to that of authentic samples. The other peaks have not yet been identified with certainty. Quczntitative estimation of desmosines. The colour intensities (peak areas) obtained with the ninhydrin-cadmium reaction of desmosine, isodesmosine, and lysinonorleucine are in a linear relationship to the
426
MOCZAR,
ROBERT,
AND
ROBERT
weight of the substances deposited up to 3 pg. The colour yield (expressed as lysine equivalents) of desmosine and isodesmosine with ninhydrin is 0.85-0.90, of lysinonorleucine 0.7, related to that of aspartic acid. Aspartic acid is convenient for comparison because it is the best separated amino acid on the electrophoretic runs under these working conditions. The same ratio is about 0.7 and 0.5, respectively, related to leucine colour. The standard error of the mean of 3 repeated determinations is of the order of 6-7s for the main peaks (aspartic acid, desmosine, isodesmosine, lysinonorleucine) and about 10% for the minor spots. DISCUSSION The reported method is simple and reliable and gives reproducible results on small samples. It can be used for the characterization of elastin samples as well as for the isolation and characterization of some of the less well known cross-linking amino acids (15). Determination of the specific activity of radioactive spots could also be carried out on elastin preparations of aortas incubated with W-lysine (17). One of the drawbacks of the method, as compared to the amino acid analyzer, is the lack of separation of desmosine and isodesmosine. Paper electrophoresis at 300 V under standard conditions appears to be sufficient to separate the desmosines from most of the other amino acids, as was shown by Blass (18). High voltage is, however, necessary for the separation of the other weakly basic amino acids participating in the cross-linking of elastin and collagen. A similar method enabling the separation and quantitative determination of hydroxylysine and its glycosides in collagen hydrolyzate was recently reported from this laboratory (19). These two micro methods together were helpful in the quantitative analytical and metabolic studies on collagen and elastin (15,17). SUMMARY A method is described for the separation and quantitative determination of the weakly basic (cross-linking) amino acids of elastin. A 6 N HCl hydrolyzate is submitted to high-voltage electrophoresis at pH 3.8. At least eight spots can be identified in the weakly basic region and quantitated by the ninhydrin-photodensitometric method. Some of these are spot 3 for desmosine + isodesmosine, and 7 for lysinonorleutine. Quantitative data given for two typical elastin preparations are in agreement with direct amino acid analysis. Preparative electrophoresis can also be carried out and used for the isolation of small quantities of the unknown spots separated by this procedure.
CROSS-LIA-KIiYG
AMINO
ACIDS
OF CLASTIN
427
ACKNOWLEDGMENTS Supported by CNRS and We would like to express Institute, Langford, England) and to Dr. J. Blass (In&it&
DGRST. our gratitude to Dr. S. M. Partridge (Meat Research for the gift of a sample of desmosine and isodesmosine Pasteur, Garches) for a sample of lysinonorleucine. REFERENCES
M., Advan. Protein Chem. 17, 227 (1962). “Structure and Function of Elastin and Collagen.” AkadCmiai Kiad6, Budapest, 1966. ROBERT, L., ROBERT, B., AND ROBERT, A. M., Exp. Gerontol. 5, 339 (1970). MANDL, I., KELLER, S., AND LEVI. M., in “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs. ed.), Vol. 1, p. 657. Academic Press, New York, 1970. COMTE, P., AND ROBERT, L., Bull. Sot. Chim. Biol. 50, 1349 (1968). GOTTE, L., STERN, P., ELSDEN, D. T.. AND PARTRIDGE, S. M., Biochem. J. 87, 344 (1963). CORBIN, K. W., Anal. Biochem. 32, 118 (1969). LEDVINA, M., AND BARTOS, F., J. Chromatogr. 31, 56 (1967). ANWAR, R. A., Can. J. Biochem. 44, 725 (1966). FRANZBLAU, C., FARIS, B., AND PAPOIOANNON, R., Biochemistry 8, 2833 (1969). STARCHER, B. C., PARTRIDGE, S. M., AKD ELSDEN, D. F., Biochemistry 6, 2425 (1967). BAILEY, A. J., PEACH, C. M., AND FOWLER, L. J., Biochemistry 117, 819 (1970). KANG, A. H., FARIS, B., AND FRANZBLAU. C., Biochem. Biophys. Res. Commun. 36, 345 (1969). LENT, R. W., SMITH, B., SALCEDO. L. L., FARIS, B., AND FRANZBLAU, C., Biochemistry 8, 2837 (1969). MOCZAR, E., ROBERT, B., DEROUETTE, J. C., AND ROBERT, L., in preparation. HEITMANN, J., BARROLIER, J., AND WATZKE, E., 2. Physiol. Chem. 309, 219 (1957). JUNQUA, S., ROBERT, A. M., AND ROBERT, L., in preparation. BLASS, J., C. R. Acad. Sci. Ser. C. 271, 94 (1970). MOCZAR, E., AND MOCZAR, M., J. Chromatogr. 51, 277 (1970).
1. PARTRIDGE, S. 2. BANGA, I., in
3. 4.
5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19.