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Valine incorporation into elastin-rich fractions from chick aorta
BIOCH1MICAET B1OPHYSICAACTA
213
BBA Report BBA 21337
Valine incorporation into elastin-rich fractions from chick aorta
R.B. RUCKER, W. GOETHLICH-RIEMANN,J. HOBE and K. DEVERS Department of Nutrition, University of California, Davis, Calif. 95616 (U.S.A. (Received May 23rd, 1972)
SUMMARY A soluble form of elastin which contains no cross-linking amino acids may be obtained from the aortas of copper-deficient chicks after formic acid extraction. The protein contains 148 residues of valine per 1000 and approximately 45 residues of lysine per 1000. Extraction of aortas with formic acid from copper-deficient and coppersupplemented chicks after intraperitoneal injection with 10/~Ci of [G-3H] valine resulted in labeled protein fractionswhich appear in part to be incorporated into more mature forms of elastin. After maximal labeling the turnover of radioactive protein in control extracts was twice as fast as that in extracts from copper-deficient aorta. The specific activity of mature elastin (alkali-insoluble) from copper-deficient chicks was 20 to 30% that from controls. However, the low value appeared to be related to the isolation procedure and not decreased synthesis. The elastin obtained from copper-deficient chicks was more easily solubilized than that from controls. In addition, the protein from copperdeficient aortas was more easily cleaved by elastase.
Mature elastin is usually resistant to common protein solvents because of crosslinks arising from select lysyl residues contained in the protein 1. Inhibition of elastin cross-linking has been achieved by nutrition copper deficiency 2, 3 and by treatment with isonicotinic acid hydrazide derivatives 4 or lathyrogens I . Recently, a soluble elastin containing no cross-links has been obtained from copper-deficient swine and chick aorta 2' 3. This protein which is isolated after formic acid extraction, fractionation by ammonium sulfate, and addition of alcohols contains approximately 40 residues of lysine per 1000. In contrast, mature elastin usually contains 5 - 9 residues of lysine per 1000 and measurable quantities of cross-linking amino acids, such as desmosine and isodesmosine (cf. refs 1-3, 7). The amino acid composition of chick soluble elastin is given in Table I. Signif-
Biochim. Biophys. Acta, 279 (1972) 213-216
214
BBA REPORT
TABLE I AMINO ACID COMPOSITION OF CHICK TROPOELASTIN The protein was isolated using the method of Sandberg et al. 2, 3. Amino acid
Residues/1000 total residues Soluble elastin Cu-defieient
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Isoleucine Leucine Tyrosine Phenylalanine Quartet-desmosine Lysine Histidine Arginine
12 7 8 13 13 148 338 180 0 148 12 46 9 12 0 48 0 3
L"
icant!y, the protein is largely hydrophobic in nature and contains approximately 14% of its total amino acid residues as valine. An examination of the rates at whieh valine is incorporated into formic acid,-, soluble protein and mature elastin is reported here in the attempt to estimate elastin synthesis hz vivo. Day-old chicks were fed copper-deficient (<2 1 ppm) and copper-supplemented (25 ppm) diets consisting primarily of skim milk 3. At 7 days chicks from both groups were injected intraperitoneally with 10/iCi of [G-3H] valine (New England Nuclear, 30 mCi/mmole). Food imake was regulated throughout the experimental period so that growth of the chicks, m both groups was equal, At 6, 1,2~ 24, 48 or 1,44 h after injection, six. chick~ from each of the two groups were killed and the aortas removed. The aortas ( 6 0 - 9 0 rag) were washed, homogenized and extracted wilh 0.02iM formic acid (48 h, 5 °C).,The spegific activity of formic acid-soluble protein and elastin in the insoluble residues was then determined. .. Radioactivity in the formic acid extracts is shown in, Fig. 1. Repeated extraction with formic acid did not resutt in a significant net increase ine.xtractable protein or radioactivity. Protein was extracted in amounts of 0.8 to 1.3rag and 0.4 to 0.6 mg per lO0:mg-. of copper-deficient and control aorta, respectively. Collagen which was estimated as Biochim. Biophys. ~cta, 279 (1972). 213 ~216
BBA REPORT
215
24
.
2O
z uJ
L6
'_o
2
25
~
12
2o
7,
0
OE
m
I
2
oo.~"2~.
4
3 DAYS
5
6
I 50
I I00 ",
MINUTES
I 150
[ 200 ,,
Fig. 1, Total radioactivity in formic acid extracts ( o - - o , -Cu; • o , + Cu) and specific activities of soluble protein ( u - - [ ] , -Cu; m - - m , + Cu) and mature elastin ( / a - - / ~ , -Cu; A - - A + Cu) from the aortas of copper-deficient and control chicks. Days indicate the time of sampling after an injection (intraperitoneally) of 10/-/Ci of [G-3H]valine into 7-day-old chicks. Fig. 2. Hydrolytic action of elastase on mature elastin from copper-deficient and control chicks. 60 mg of elastin were incubated in the presence of 20 units of elastase for the times indicated at pH 8.8. The rate of hydrolysis of copper-deficient (© . o) and control ( o - - e ) elastin preparations was measured after reaction of liberated products with ninhydrin (cf. ref. 7).
hydroxyproline 6 amounted to only 10 to 15% of the total protein in extracts. Since elastin contains 6 - 8 times more valine than collagen and other proteins, changes in specific activity were assumed to be due primarily to changes in the disappearance of elastin-like protein. After maximal labeling with [G-3H] valine, 24 h were required for a 50% reduction in the specific activity of the labeled protein extracted from copperdeficient chick aortas compared to only 12 h of that in controls. These values appear reasonable in that mature elastin in the normal chick aorta (7 to 14 days old) increased approximately 0.4 rag/day per 100 mg of tissue during this period. There were no differences in the labeling of plasma proteins when copper-deficient and control chicks were compared during the experimental periods. Mature elastin was defined as that protein which resisted extraction by alkali treatment s. With respect to incorporation of [G-3H] valine into this protein in aortas from copper-deficient chicks (Fig, t ), it appeared thnt radioactivity was incorporated into only partially cross-linked mature elastin. The specific activity of mature elastin fractions from copper-deficient chicks was 20 to 30% that of control material indicating most of the newly formeu m~ttutc elasun was solubihzed by alkali treatment. As shown in Fig. 2, the elastin from copper-deficient chicks was also more susceptible to the hydrolytic action of elastase. Elastase activity was determined by the methods of Schneider et al. 7. The data suggest that the decrease in insoluble elastin usually associated with copper deficiency 3 Bio chim. B i o p h y s . A cta, 2 7 9 ( 19 7 2 ) 213 -- 216
216
F~BAREPORT
may be due not only to a decrease in cross-linking, but also its susceptability to degradation when less cross-linked. The effect of elastase on soluble forms of elastin is presently being investigated. Supported in part by a grant from the Nutrition Foundation. REFERENCES 1 2 3 4 5 6 7
K.A. Piez, Annu. Rev. Biochem., 37 (1968) 547. L.B. Sandberg, R.D. Zeikus and I.M. Coltrain, Biochim. Biophys. Acta, 236 (1971) 542. R.B. Rucker and W. Goethlich-Riemann, J. Nutr., 102 (1972) 563. R.B. Rucker and B.L. O'Dell, Biochim. Biophys. Acta, 222 (1970) 527. A.I. Lansing, T.B. Rosenthal, M. Alex, E.W. Dempsey, Anat. Rec., 114 (1952) 555. J.F. Woessner, Jr.,Arch. Biochem. Biophys., 93 (1961) 440. I.J. Schneider, S. Tindel, D. Shapiro and D. State, Z Lab. Clin. Med., 60 (1962) 514.
Biochim. Biophys. Acta, 279 (1972) 213-216
213
BBA Report BBA 21337
Valine incorporation into elastin-rich fractions from chick aorta
R.B. RUCKER, W. GOETHLICH-RIEMANN,J. HOBE and K. DEVERS Department of Nutrition, University of California, Davis, Calif. 95616 (U.S.A. (Received May 23rd, 1972)
SUMMARY A soluble form of elastin which contains no cross-linking amino acids may be obtained from the aortas of copper-deficient chicks after formic acid extraction. The protein contains 148 residues of valine per 1000 and approximately 45 residues of lysine per 1000. Extraction of aortas with formic acid from copper-deficient and coppersupplemented chicks after intraperitoneal injection with 10/~Ci of [G-3H] valine resulted in labeled protein fractionswhich appear in part to be incorporated into more mature forms of elastin. After maximal labeling the turnover of radioactive protein in control extracts was twice as fast as that in extracts from copper-deficient aorta. The specific activity of mature elastin (alkali-insoluble) from copper-deficient chicks was 20 to 30% that from controls. However, the low value appeared to be related to the isolation procedure and not decreased synthesis. The elastin obtained from copper-deficient chicks was more easily solubilized than that from controls. In addition, the protein from copperdeficient aortas was more easily cleaved by elastase.
Mature elastin is usually resistant to common protein solvents because of crosslinks arising from select lysyl residues contained in the protein 1. Inhibition of elastin cross-linking has been achieved by nutrition copper deficiency 2, 3 and by treatment with isonicotinic acid hydrazide derivatives 4 or lathyrogens I . Recently, a soluble elastin containing no cross-links has been obtained from copper-deficient swine and chick aorta 2' 3. This protein which is isolated after formic acid extraction, fractionation by ammonium sulfate, and addition of alcohols contains approximately 40 residues of lysine per 1000. In contrast, mature elastin usually contains 5 - 9 residues of lysine per 1000 and measurable quantities of cross-linking amino acids, such as desmosine and isodesmosine (cf. refs 1-3, 7). The amino acid composition of chick soluble elastin is given in Table I. Signif-
Biochim. Biophys. Acta, 279 (1972) 213-216
214
BBA REPORT
TABLE I AMINO ACID COMPOSITION OF CHICK TROPOELASTIN The protein was isolated using the method of Sandberg et al. 2, 3. Amino acid
Residues/1000 total residues Soluble elastin Cu-defieient
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Isoleucine Leucine Tyrosine Phenylalanine Quartet-desmosine Lysine Histidine Arginine
12 7 8 13 13 148 338 180 0 148 12 46 9 12 0 48 0 3
L"
icant!y, the protein is largely hydrophobic in nature and contains approximately 14% of its total amino acid residues as valine. An examination of the rates at whieh valine is incorporated into formic acid,-, soluble protein and mature elastin is reported here in the attempt to estimate elastin synthesis hz vivo. Day-old chicks were fed copper-deficient (<2 1 ppm) and copper-supplemented (25 ppm) diets consisting primarily of skim milk 3. At 7 days chicks from both groups were injected intraperitoneally with 10/iCi of [G-3H] valine (New England Nuclear, 30 mCi/mmole). Food imake was regulated throughout the experimental period so that growth of the chicks, m both groups was equal, At 6, 1,2~ 24, 48 or 1,44 h after injection, six. chick~ from each of the two groups were killed and the aortas removed. The aortas ( 6 0 - 9 0 rag) were washed, homogenized and extracted wilh 0.02iM formic acid (48 h, 5 °C).,The spegific activity of formic acid-soluble protein and elastin in the insoluble residues was then determined. .. Radioactivity in the formic acid extracts is shown in, Fig. 1. Repeated extraction with formic acid did not resutt in a significant net increase ine.xtractable protein or radioactivity. Protein was extracted in amounts of 0.8 to 1.3rag and 0.4 to 0.6 mg per lO0:mg-. of copper-deficient and control aorta, respectively. Collagen which was estimated as Biochim. Biophys. ~cta, 279 (1972). 213 ~216
BBA REPORT
215
24
.
2O
z uJ
L6
'_o
2
25
~
12
2o
7,
0
OE
m
I
2
oo.~"2~.
4
3 DAYS
5
6
I 50
I I00 ",
MINUTES
I 150
[ 200 ,,
Fig. 1, Total radioactivity in formic acid extracts ( o - - o , -Cu; • o , + Cu) and specific activities of soluble protein ( u - - [ ] , -Cu; m - - m , + Cu) and mature elastin ( / a - - / ~ , -Cu; A - - A + Cu) from the aortas of copper-deficient and control chicks. Days indicate the time of sampling after an injection (intraperitoneally) of 10/-/Ci of [G-3H]valine into 7-day-old chicks. Fig. 2. Hydrolytic action of elastase on mature elastin from copper-deficient and control chicks. 60 mg of elastin were incubated in the presence of 20 units of elastase for the times indicated at pH 8.8. The rate of hydrolysis of copper-deficient (© . o) and control ( o - - e ) elastin preparations was measured after reaction of liberated products with ninhydrin (cf. ref. 7).
hydroxyproline 6 amounted to only 10 to 15% of the total protein in extracts. Since elastin contains 6 - 8 times more valine than collagen and other proteins, changes in specific activity were assumed to be due primarily to changes in the disappearance of elastin-like protein. After maximal labeling with [G-3H] valine, 24 h were required for a 50% reduction in the specific activity of the labeled protein extracted from copperdeficient chick aortas compared to only 12 h of that in controls. These values appear reasonable in that mature elastin in the normal chick aorta (7 to 14 days old) increased approximately 0.4 rag/day per 100 mg of tissue during this period. There were no differences in the labeling of plasma proteins when copper-deficient and control chicks were compared during the experimental periods. Mature elastin was defined as that protein which resisted extraction by alkali treatment s. With respect to incorporation of [G-3H] valine into this protein in aortas from copper-deficient chicks (Fig, t ), it appeared thnt radioactivity was incorporated into only partially cross-linked mature elastin. The specific activity of mature elastin fractions from copper-deficient chicks was 20 to 30% that of control material indicating most of the newly formeu m~ttutc elasun was solubihzed by alkali treatment. As shown in Fig. 2, the elastin from copper-deficient chicks was also more susceptible to the hydrolytic action of elastase. Elastase activity was determined by the methods of Schneider et al. 7. The data suggest that the decrease in insoluble elastin usually associated with copper deficiency 3 Bio chim. B i o p h y s . A cta, 2 7 9 ( 19 7 2 ) 213 -- 216
216
F~BAREPORT
may be due not only to a decrease in cross-linking, but also its susceptability to degradation when less cross-linked. The effect of elastase on soluble forms of elastin is presently being investigated. Supported in part by a grant from the Nutrition Foundation. REFERENCES 1 2 3 4 5 6 7
K.A. Piez, Annu. Rev. Biochem., 37 (1968) 547. L.B. Sandberg, R.D. Zeikus and I.M. Coltrain, Biochim. Biophys. Acta, 236 (1971) 542. R.B. Rucker and W. Goethlich-Riemann, J. Nutr., 102 (1972) 563. R.B. Rucker and B.L. O'Dell, Biochim. Biophys. Acta, 222 (1970) 527. A.I. Lansing, T.B. Rosenthal, M. Alex, E.W. Dempsey, Anat. Rec., 114 (1952) 555. J.F. Woessner, Jr.,Arch. Biochem. Biophys., 93 (1961) 440. I.J. Schneider, S. Tindel, D. Shapiro and D. State, Z Lab. Clin. Med., 60 (1962) 514.
Biochim. Biophys. Acta, 279 (1972) 213-216