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Binding of manganese and transferrin in rat serum
300
SHORT COMMUNICATIONS
Sulphate, phosphate and citrate are known to increase the partition coefficient of negatively charged proteins 2, in agreement with the proposed interaction between dextran and the anions. This work has been supported by grants from STU-Swedish Board for Technical Development. I wish to thank Professor Per-~ke Albertsson for his interest and encouragement during the course of this work.
Department of Biochemistry University of Umed, Umed (Sweden)
GOTE .JOHANSSON
I P . . ~ . ALBERTSSON, Partition of Cell Particles and Macromolecules, A l m q v i s t a n d Wiksells, S t o c k h o l m , Wiley, New York, I96O. 2 P. ~. ALBERTSSON, Advances in Protein Chemistry, Vol. 24, A c a d e m i c Press, New York, 197 o, p. 3o9 . 3 P-,Ok. ALBERTSSON, Partition of Cell Particles and Macromolecules, A l m q v i s t a n d Wiksells, S t o c k h o l m , Wiley, N e w York, I96O. pp. 28-34. 4 P.-~. ALBERTSSON, Partition of Cell Particles and Macromolecules, A l m q v i s t a n d Wiksells, S t o c k h o l m , Wiley, New York, I96O, p. 49. 5 E. PRICE, in J. J. LAGOWSKI, The Chemistry of Non-Aqueous Solvents, Vol. l, A c a d e m i c Press, New York, 1966 , p. 67. 6 A. J. PARKER, Quart. Rev. London, 16 (1902) 163. 7 F. A. COTTON AND G. WILKINSON, Advanced Inorganic Chemistry, Wiley, New York, 1966, pp. 422, 423. S G. JOHANSSON, u n d e r publication. 9 H. MAHLER AND E. CORDES, Biological Chemistry, H a r p e r a n d Row, New York, I966, p. 54io H. WALTER, R. GARZA AND R. P. COYLE, Biochim. Biophys. Acta, 156 (1968) 4o9.
Received June 22nd, 197o
Biochim. Biophys. Acta, 22I (197o) 387-39°
BBA 33248 Binding of manganese and transferrin in rat serum Previous investigators z working on the transport of manganese in blood have suggested the presence of a protein other than transferrin that binds manganese. In contrast, PESENDORFER et al3 determined that human transferrin does bind manganese in serum. Also PANIC3 demonstrated that manganese, when incubated with bovine serum, binds to transferrin. In the course of studies investigating the mechanism by which a choline-deficient diet lowers liver manganesO, it became necessary to study the binding of manganese by rat serum proteins. The results of these studies indicate that in the rat, transferrin may be the serum protein which binds manganese.
Materials and methods Blood samples. Male albino rats (Sprague-Dawley strain) weighing 300-350 g were used in this study. The animals were fed a standard rat diet (Purina Laboratory Rat Chow) or a choline-deficient rat diet (General Biochemicals) for 21 days. Tap
Biochim. Biophys. dcta, 22I (197o) 390 393
SHORT COMMUNICATIONS
391
water and diet were consumed ad libitum by the rats which were housed in normal stainless steel cages. Rats were placed under ether anesthesia and exsanguinated b y drawing blood from the abdominal aorta. Radioactive labeling of serum. Serum prepared from the blood was incubated for i h at 37 ° with carrier-free S4MnC12 (Mallinkrodt Nuclear, Orlando, Florida, radiopurity 99%) in the ratio of o.I #C to each ml of serum. In the experiments involving manganese and iron the same amount of radiomanganese was used along with o.I #C of carrier-free 59re citrate (Squibb Pharmaceutical Co., East Brunswick, N.J.) for each ml of serum. Following incubation the serum was dialyzed against physiological saline for i h and separated into its constituent proteins by block electrophoresis. Pevikon-Geon block electrophoresis. The polymer was prepared with equal quantities of Pevikon C-87o (Stockholms Superfosfat Fabriks, A-B., Stockholm) and Geon 427 (Goodrich Chem. Co., Akron, Ohio) as a slurry in a barbital buffer, pH 8.6, ionic strength 0.06 (ref. 5). Each block, supported by a plexiglass tray (io cm × I cm), was divided into three compartments to accomodate three 3-ml serum samples. Electrophoresis was performed at 4 ° with 30 mA for 18 h, giving a total anodal migration of approximately 27 cm. Each block was cut into I-cm strips. The protein in each fraction was eluted with o.14 M NaCl and diluted to 20 ml. Absorbance at 280 nm was determined with a Beckman DU-2 Spectrophotometer. Radioactivity of 54Mn and 5~Fe in counts per min was measured with a Baird-Atomic automatic g a m m a counter. The level of radioactivity in each sample after electrophoresis was plotted immediately under the protein profile indicating the relative position of manganese and iron. Samples containing peak radioactivity were subjected to immunoelectrophoresis and double radial immunodiffusion in gel to determine the specific type of protein present in these samples. Immunoelectrophoresis. Immunoelectrophoresis was performed by the method of CAWLEY et al. 6 using 1% agarose (Mann Research Laboratories, New York, N.Y.) in barbital buffer p H 8.6, ionic strength 0.03, supported on 33 m m polyester photographic film (Dupont Cronar, P. 4oB). 5-/A samples were applied to the wells, and electrophoresis was performed in barbital buffer pH 8.6, ionic strength 0.06 at 6 mA per strip for 9 ° rain. 5o #1 of rat antiserum (Hyland Laboratories, Los Angeles, Calif.) was applied to the trough, and diffusion was allowed to take place for 24 h at room temperature. Excess antiserum was removed by washing in saline for 24 h and in distilled water for 24 h. The strips were dried and stained with amido Schwartz loB. Electrofocusing. The electrofocusing technique of VESTERBERG AND SVENSSON7 was used in this study, employing an ampholite p H gradient from p H 5 to 8. Results and discussion BARAK et al. 4 reported that under the influence of a choline deficiency, rat liver manganese is decreased. Recently more extensive work has confirmed this earlier finding and has indicated changes in liver manganese 5 days after administration of choline-deficient diets (unpublished observations). With this in mind, it was felt pertinent to determine if there is a failure in the transport of serum manganese which would contribute to a deficiency of liver manganese. The experiments that are described were perfolmed on serum from choline-deficient animals as well as from normal control animals. Biochim. Biophys. Acta, 221 (197o) 39o-393
392
SHORT COMMUNICATIONS
The separation of a4Mn-labeled rat serum by block electrophoresis is shown in Fig. I. Tube o is the point of application of the serum, and the peak about tube 26 represents the albumin fraction. The majority of the nondialyzable radioactivity was confined to tubes 15, 16 and 17 which corresponds to a sizeable protein peak. A minor amount of radioactivity was found in the tubes containing the albumin traction. Immunoelectrophoresis of the eluent in tube 16 demonstrated the presence of a single
2.2
,22 0 0 0
2.0
20 0 0 0
1.8
18 0 0 0
1.6
16 0 0 0
1.4
14 0 0 0
1.2
12 0 0 0
z
i
1.0
:3B
/
i.54Mn
I0 0 0 0
0.8
8 000
/
0.6
_~ ~ m
6 000
0.4
4 000
0.2
2 000
J " e " e ' ~ '''''e ~ ''e'e*~ ~,,m'•
0 0
4
8
12
I
16
I~
20
,
24
?"!
0
28
TUBE NUMBER
Fig. I. Separation of ~q~In-labeled rat serum by block electrophoresis. O " " O , P a t t e r n of radioactive 54Mn in t h e eluent fractions; 0 - - 0 , absorbance of the eluent fractions.
protein p~ecipitin band. For comparison, normal rat serum was analyzed simultaneously. Tile protein of tube 16 corresponds to transferrin of normal rat serum. In order to confirm the above results, another method of analysis was utilized in the study. The eluents of tubes 15, 16 and 17 from block electrophoresis were pooled and introduced at the top (cathode) of a column prepared for electrofocusing. The elution pattern obtained after 114 h of continuous current was obtained for both the protein concentration and the radioactivity. The radioactivity of the 54Mn was found in high concentration in one protein peak. The isoelectric point of the protein as determined by measuring the pH of the peak tube was 5-7. The isoelectric point of human transferrin is 5.9 (ref. S). A discrepancy between these two values may be explained on the basis of species difference and/or on the presence of macromolecules in the solution which make it difficult to ascertain accurately the pH of protein solutions:L Immunoelectrophoresis of the eluent of Tube 4 1 showed the presence of a single protein precipitin band which corresponds to transferrin of whole rat serum. Fig. 2 illustrates results gained from a typical double-labeling experiment with both 54Mn and 59Fe. It is seen that in the rat a single protein binds the majority of Biochim. Biophys..4cta, 221 (197o) 39 ° 393
393
SHORT COMMUNICATIONS
70 0 0 0 60 000
1.2
'~I.0
e
0.8
•
5O 0 0 0
40 000
,t
30 000
0.4
0
5'Fe--/
i
l
,
0
4
8
I
i
"t,~o,I~
16
m
I0 0 0 0
~ 5'lMn 12
"v
20 000 ~
\~
0"2 /
0
i
I
20
24
0 28
TUBE NUMBER
Fig. 2. Block electrophoresis of r a t s e r u m i n c u b a t e d w i t h 54Mn a n d SgFe. O - - O , a b s o r b a n c e o f e l u e n t fractions; • ..... 0 , p a t t e r n of r a d i o a c t i v e 59Fe in e l u e n t fractions; o m m m o , p a t t e r n of r a d i o a c t i v e 54Mn in e l u e n t fractions.
both metals. Immunoelectrophoresis studies have shown this protein to be the same as that binding manganese in the initial experiments. The results of these experiments with choline-deficient serum are identical with those conducted with normal serum and show that the decrease seen in liver manganese in a choline deficiency is likely not due to a defect in the manganese transport system of plasma. A major finding of these experiments indicates that transferrin is the protein that binds manganese in rat serum.
Medical Research Laboratory, Veterans Administration Hospital, Omaha, Nebraska 68105 (U.S.A.)
R O B E R T C. K E E F E R ANTHONY J. BARAK JOHN D. BOYETT
I G. C. COTZIAS, in C. L. COMAR AND F. BRONNER, Mineral Metabolism, Vol. 2, A c a d e m i c Press, New York, 1962, p. 4o4 . 2 V. PESENDORFER, H. FRISCHAUF AND F. WEWALKA, Protides Biol. Fluids, Proc. Colloq., 14 (1966) 193. 3 B. PANIC, Acta Vet. Scand., 8 (1967) 228. 4 A. J. BARAK, H. C. BECKENHAUER AND F. J. KERRIGAN, Gut, 8 (1967) 454" 5 J. L. FAHEY AND C. McLAUGHLIN, J. Immunol., 91 (1963) 484 . 6 L. P. CAWLEY, L. EBERHARDT AND D. SCHNEIDER, J. Lab. Clin. Med., 65 (1965) 324. 7 0 . VESTERBERG AND H. SVENSSON, Acta Chem. Scand., 20 (1966) 820. 8 H. E. SCHULTZE AND J . F . HEREMANS, Molecular Biology of Human Proteins, Elsevier, N e w York, 1966, p. 179. 9 C. TANFORD, in C. B. ANFINSEN, JR., K. BAILEY, M. C. ANSON AND J. T. EDSALL, Advances in Protein Chemistry, Vol. 17, A c a d e m i c Press, New York, 1962, p. 69.
Received July I6th, 197o Biochim. Biophys. Acta, 221 (197o) 390-393
SHORT COMMUNICATIONS
Sulphate, phosphate and citrate are known to increase the partition coefficient of negatively charged proteins 2, in agreement with the proposed interaction between dextran and the anions. This work has been supported by grants from STU-Swedish Board for Technical Development. I wish to thank Professor Per-~ke Albertsson for his interest and encouragement during the course of this work.
Department of Biochemistry University of Umed, Umed (Sweden)
GOTE .JOHANSSON
I P . . ~ . ALBERTSSON, Partition of Cell Particles and Macromolecules, A l m q v i s t a n d Wiksells, S t o c k h o l m , Wiley, New York, I96O. 2 P. ~. ALBERTSSON, Advances in Protein Chemistry, Vol. 24, A c a d e m i c Press, New York, 197 o, p. 3o9 . 3 P-,Ok. ALBERTSSON, Partition of Cell Particles and Macromolecules, A l m q v i s t a n d Wiksells, S t o c k h o l m , Wiley, N e w York, I96O. pp. 28-34. 4 P.-~. ALBERTSSON, Partition of Cell Particles and Macromolecules, A l m q v i s t a n d Wiksells, S t o c k h o l m , Wiley, New York, I96O, p. 49. 5 E. PRICE, in J. J. LAGOWSKI, The Chemistry of Non-Aqueous Solvents, Vol. l, A c a d e m i c Press, New York, 1966 , p. 67. 6 A. J. PARKER, Quart. Rev. London, 16 (1902) 163. 7 F. A. COTTON AND G. WILKINSON, Advanced Inorganic Chemistry, Wiley, New York, 1966, pp. 422, 423. S G. JOHANSSON, u n d e r publication. 9 H. MAHLER AND E. CORDES, Biological Chemistry, H a r p e r a n d Row, New York, I966, p. 54io H. WALTER, R. GARZA AND R. P. COYLE, Biochim. Biophys. Acta, 156 (1968) 4o9.
Received June 22nd, 197o
Biochim. Biophys. Acta, 22I (197o) 387-39°
BBA 33248 Binding of manganese and transferrin in rat serum Previous investigators z working on the transport of manganese in blood have suggested the presence of a protein other than transferrin that binds manganese. In contrast, PESENDORFER et al3 determined that human transferrin does bind manganese in serum. Also PANIC3 demonstrated that manganese, when incubated with bovine serum, binds to transferrin. In the course of studies investigating the mechanism by which a choline-deficient diet lowers liver manganesO, it became necessary to study the binding of manganese by rat serum proteins. The results of these studies indicate that in the rat, transferrin may be the serum protein which binds manganese.
Materials and methods Blood samples. Male albino rats (Sprague-Dawley strain) weighing 300-350 g were used in this study. The animals were fed a standard rat diet (Purina Laboratory Rat Chow) or a choline-deficient rat diet (General Biochemicals) for 21 days. Tap
Biochim. Biophys. dcta, 22I (197o) 390 393
SHORT COMMUNICATIONS
391
water and diet were consumed ad libitum by the rats which were housed in normal stainless steel cages. Rats were placed under ether anesthesia and exsanguinated b y drawing blood from the abdominal aorta. Radioactive labeling of serum. Serum prepared from the blood was incubated for i h at 37 ° with carrier-free S4MnC12 (Mallinkrodt Nuclear, Orlando, Florida, radiopurity 99%) in the ratio of o.I #C to each ml of serum. In the experiments involving manganese and iron the same amount of radiomanganese was used along with o.I #C of carrier-free 59re citrate (Squibb Pharmaceutical Co., East Brunswick, N.J.) for each ml of serum. Following incubation the serum was dialyzed against physiological saline for i h and separated into its constituent proteins by block electrophoresis. Pevikon-Geon block electrophoresis. The polymer was prepared with equal quantities of Pevikon C-87o (Stockholms Superfosfat Fabriks, A-B., Stockholm) and Geon 427 (Goodrich Chem. Co., Akron, Ohio) as a slurry in a barbital buffer, pH 8.6, ionic strength 0.06 (ref. 5). Each block, supported by a plexiglass tray (io cm × I cm), was divided into three compartments to accomodate three 3-ml serum samples. Electrophoresis was performed at 4 ° with 30 mA for 18 h, giving a total anodal migration of approximately 27 cm. Each block was cut into I-cm strips. The protein in each fraction was eluted with o.14 M NaCl and diluted to 20 ml. Absorbance at 280 nm was determined with a Beckman DU-2 Spectrophotometer. Radioactivity of 54Mn and 5~Fe in counts per min was measured with a Baird-Atomic automatic g a m m a counter. The level of radioactivity in each sample after electrophoresis was plotted immediately under the protein profile indicating the relative position of manganese and iron. Samples containing peak radioactivity were subjected to immunoelectrophoresis and double radial immunodiffusion in gel to determine the specific type of protein present in these samples. Immunoelectrophoresis. Immunoelectrophoresis was performed by the method of CAWLEY et al. 6 using 1% agarose (Mann Research Laboratories, New York, N.Y.) in barbital buffer p H 8.6, ionic strength 0.03, supported on 33 m m polyester photographic film (Dupont Cronar, P. 4oB). 5-/A samples were applied to the wells, and electrophoresis was performed in barbital buffer pH 8.6, ionic strength 0.06 at 6 mA per strip for 9 ° rain. 5o #1 of rat antiserum (Hyland Laboratories, Los Angeles, Calif.) was applied to the trough, and diffusion was allowed to take place for 24 h at room temperature. Excess antiserum was removed by washing in saline for 24 h and in distilled water for 24 h. The strips were dried and stained with amido Schwartz loB. Electrofocusing. The electrofocusing technique of VESTERBERG AND SVENSSON7 was used in this study, employing an ampholite p H gradient from p H 5 to 8. Results and discussion BARAK et al. 4 reported that under the influence of a choline deficiency, rat liver manganese is decreased. Recently more extensive work has confirmed this earlier finding and has indicated changes in liver manganese 5 days after administration of choline-deficient diets (unpublished observations). With this in mind, it was felt pertinent to determine if there is a failure in the transport of serum manganese which would contribute to a deficiency of liver manganese. The experiments that are described were perfolmed on serum from choline-deficient animals as well as from normal control animals. Biochim. Biophys. Acta, 221 (197o) 39o-393
392
SHORT COMMUNICATIONS
The separation of a4Mn-labeled rat serum by block electrophoresis is shown in Fig. I. Tube o is the point of application of the serum, and the peak about tube 26 represents the albumin fraction. The majority of the nondialyzable radioactivity was confined to tubes 15, 16 and 17 which corresponds to a sizeable protein peak. A minor amount of radioactivity was found in the tubes containing the albumin traction. Immunoelectrophoresis of the eluent in tube 16 demonstrated the presence of a single
2.2
,22 0 0 0
2.0
20 0 0 0
1.8
18 0 0 0
1.6
16 0 0 0
1.4
14 0 0 0
1.2
12 0 0 0
z
i
1.0
:3B
/
i.54Mn
I0 0 0 0
0.8
8 000
/
0.6
_~ ~ m
6 000
0.4
4 000
0.2
2 000
J " e " e ' ~ '''''e ~ ''e'e*~ ~,,m'•
0 0
4
8
12
I
16
I~
20
,
24
?"!
0
28
TUBE NUMBER
Fig. I. Separation of ~q~In-labeled rat serum by block electrophoresis. O " " O , P a t t e r n of radioactive 54Mn in t h e eluent fractions; 0 - - 0 , absorbance of the eluent fractions.
protein p~ecipitin band. For comparison, normal rat serum was analyzed simultaneously. Tile protein of tube 16 corresponds to transferrin of normal rat serum. In order to confirm the above results, another method of analysis was utilized in the study. The eluents of tubes 15, 16 and 17 from block electrophoresis were pooled and introduced at the top (cathode) of a column prepared for electrofocusing. The elution pattern obtained after 114 h of continuous current was obtained for both the protein concentration and the radioactivity. The radioactivity of the 54Mn was found in high concentration in one protein peak. The isoelectric point of the protein as determined by measuring the pH of the peak tube was 5-7. The isoelectric point of human transferrin is 5.9 (ref. S). A discrepancy between these two values may be explained on the basis of species difference and/or on the presence of macromolecules in the solution which make it difficult to ascertain accurately the pH of protein solutions:L Immunoelectrophoresis of the eluent of Tube 4 1 showed the presence of a single protein precipitin band which corresponds to transferrin of whole rat serum. Fig. 2 illustrates results gained from a typical double-labeling experiment with both 54Mn and 59Fe. It is seen that in the rat a single protein binds the majority of Biochim. Biophys..4cta, 221 (197o) 39 ° 393
393
SHORT COMMUNICATIONS
70 0 0 0 60 000
1.2
'~I.0
e
0.8
•
5O 0 0 0
40 000
,t
30 000
0.4
0
5'Fe--/
i
l
,
0
4
8
I
i
"t,~o,I~
16
m
I0 0 0 0
~ 5'lMn 12
"v
20 000 ~
\~
0"2 /
0
i
I
20
24
0 28
TUBE NUMBER
Fig. 2. Block electrophoresis of r a t s e r u m i n c u b a t e d w i t h 54Mn a n d SgFe. O - - O , a b s o r b a n c e o f e l u e n t fractions; • ..... 0 , p a t t e r n of r a d i o a c t i v e 59Fe in e l u e n t fractions; o m m m o , p a t t e r n of r a d i o a c t i v e 54Mn in e l u e n t fractions.
both metals. Immunoelectrophoresis studies have shown this protein to be the same as that binding manganese in the initial experiments. The results of these experiments with choline-deficient serum are identical with those conducted with normal serum and show that the decrease seen in liver manganese in a choline deficiency is likely not due to a defect in the manganese transport system of plasma. A major finding of these experiments indicates that transferrin is the protein that binds manganese in rat serum.
Medical Research Laboratory, Veterans Administration Hospital, Omaha, Nebraska 68105 (U.S.A.)
R O B E R T C. K E E F E R ANTHONY J. BARAK JOHN D. BOYETT
I G. C. COTZIAS, in C. L. COMAR AND F. BRONNER, Mineral Metabolism, Vol. 2, A c a d e m i c Press, New York, 1962, p. 4o4 . 2 V. PESENDORFER, H. FRISCHAUF AND F. WEWALKA, Protides Biol. Fluids, Proc. Colloq., 14 (1966) 193. 3 B. PANIC, Acta Vet. Scand., 8 (1967) 228. 4 A. J. BARAK, H. C. BECKENHAUER AND F. J. KERRIGAN, Gut, 8 (1967) 454" 5 J. L. FAHEY AND C. McLAUGHLIN, J. Immunol., 91 (1963) 484 . 6 L. P. CAWLEY, L. EBERHARDT AND D. SCHNEIDER, J. Lab. Clin. Med., 65 (1965) 324. 7 0 . VESTERBERG AND H. SVENSSON, Acta Chem. Scand., 20 (1966) 820. 8 H. E. SCHULTZE AND J . F . HEREMANS, Molecular Biology of Human Proteins, Elsevier, N e w York, 1966, p. 179. 9 C. TANFORD, in C. B. ANFINSEN, JR., K. BAILEY, M. C. ANSON AND J. T. EDSALL, Advances in Protein Chemistry, Vol. 17, A c a d e m i c Press, New York, 1962, p. 69.
Received July I6th, 197o Biochim. Biophys. Acta, 221 (197o) 390-393