CONFORMATIONAL
VARIATION IN A
PLASMA
Hood, L., Lackland, J., and Krause, R . M. (1970), Fed. Proc., Fed. Amer. SOC.Exp. Biol. 29,438. Jaton, J. C., Waterfield, M. D., Margolies, M. N., Bloch, K. J., and Haber, E. (1971), Biochemistry 10, 1583. Jaton, J. C.?Waterfield, M. D., Margolies, M. N., and Haber, E. (1970), Proc. Nut. Acad. Sci. U . S . 66,969. Kimball, J. W., Pappenheimer, A. M., Jr., Jaton, J. C. (1971), J. Immunol. 106,1177. Klotz, I. M. (1967), Methods Enzymol. 11, 576. Krause, R. M. (1970), Fed. Proc., Fed. Amer. SOC.Exp. Biol. 29, 59. Lamm, M. E., and Frangione, B. (1972), Biochem. J. 128,1357. Levy, A. B., and Sober, H. A. (1960), Proc. SOC.Exp. Biol. 103,250. Milstein, C. (1967), Nature (London) 216, 330. Milstein, C., Clegg, J. B., and Jarvis, J. M. (1968), Biochem. J . 110,631. Niall, H. D., and Potts, J. T., Jr. (1970), in Peptides: Chemistry and Biochemistry, Weinstein, B., and Lande, S., Ed., New York, N. Y., Marcel Dekker, p 215.
LIPOPROTEIN
Pincus, J. H.,Jaton, J. C., Bloch, K. J., and Haber, E. (1970a), J. Immunol. 104, 1143. Pincus, J. H., Jaton, J. C., Bloch, K. J., and Haber, E. (1970b), J. Immunol. 104, 1149. Pisano, J. J. and Bronzert, T. J. (1969), J. Biol. Chen?. 244, 5597. Poljak, I 0. In the Pearson product-moment correlation, the correlation coefficient, r = -0.178, which value signifies no correlation (Fisher and Yates, 1963). The highestfn (=0.74) was found for the a-2 globulin sample with the lowest concentration ( = 4 . 7 pg/ml) (Figure 4). For the matching control sample the concentration = 6.1 pg/ml; this sample was clearly nonhelical. Among the patient 01-2 globulin samples with fH > 0, the ten lowest in mncentration had a mean concentration = 14.8 fig/ml, and the mean f H = 0.33. For the nonhelical patient samples, the mean concentration = 21 pg/ml. For the protein samples from the control subjects, the mean concentration = 33 fig/ml. The conformations of these protein samples were clearly independent of concentration.
Bar graphs of results of conformation studies on 47 a-2 globulin samples from 1 3 patients, Each rectangle depicts the results on one a-2 globulin sample. Numbers depict average.h (from bo, [ m ' l ~ [rn'lm, ~, and [O'IS., when measured). Shading depicts nonhelical conformations. FIGURE 9:
Discussion Similarifies and Differences of a-2 Globulin Fractions. The a-2 globulin samples from the control subjects and from the patients are very similar or identical in many ways. These similarities are summarized in Table I. With all of these similarities, it seems that these a-2 globulins are the same protein.
I CONFORMATION Sample Number
Control
Ioljl
.>....: 3
H
I
I
l 1 1Bar graphs of results of conformation studies on 31 a-2 globulin samples from 17 control subjects. Numbers and shadings have same significance as in Figure 9. FIGURE IO:
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TABLE I : Comparisons of Properties of CY-2Globulin Fractions from Control Subjects and from Patients.
Differences in Activities of Plasma a-2 Globulin Samples from Control Subjects and from Patients.
TABLE 11:
-.. -
Property Curtain electrophoresis fraction Column chromatography fraction Sucrose gradient fraction RE’in disc electrophoresis Lipid moiety : content
From Control Subject Tube 18
From Patient
-
Tube 18
31
31
1 0.53 80
1 0.53 80
High Present Present Present 20
High Present Present Present 20
14.5 23.2 3.5 4.7 6.4 4.9 8.6 8.9 7.3 Tr 12.3 3.5 4.9 310 + 53
14.8 21.4 3.6 4.8 6.6 5.2 8.8 9.1 6.8 11.8 3.2 4.6 293 + 50
Near 400,000
Near 400,000
( %)
Cholesterol Linoleic acid Oleic acid Stearic acid Protein moiety : content ( %I
+
LYS Arg (mol %) Asp Glu His Thr Ser Pro GlY Ala Val Met Ile Leu
+
+
TYr Phe Plasma level (pg/250 ml of plasma) Molecular weight
Activity
Tr
-
.
Glu transport (pg/ml of erythrocytes per hr) Trp transport (dpm X 10-3/g of erythrocytes per hr) Serotonin in brain tissue after incubation (ng/g) MezTrm in brain tissue after incubation (ng/g) Lactate :pyruvate in chicken erythrocyte incubation Variation in lactate : pyruvate Lactate :pyruvate after exercise Insulin effects AATP synthesis’ (dpm x 10-3/g per min) AFru + Fru-1,6-P2 (dpm X 10-3/g per min) AEmbden-Meyerhof pathway (dpm x 10-3/g of tissue) AHMP pathway (dpm X 10-3/g of tissue) Acetate + lactate (dpm X mol) NADH 0 2 -+ NAD’ H20 normal) NADPH 0: -+ NADP+ H 2 0 (% normal) Rope-climbing time of rats‘ (min-sec) Self-stimulation of rats’ (Abar presses/min)
+ +
+
(z
+
~-
From Control Subject
-____
From Patient
-._ -- - -
343
221
93
142
8.51
10 07
1.07
1 81
4.9
8 7
110.3 13.7
123 7 80 0
+440 +340
-
- 260 - 360 -2 69
$0.86 -6.5
+12 0
1404
780
89.6
86 9
94.6
82 0
32 3
957
+4
- 36 -~
These activities were measured in cicn. The other activities were from in citro tests. a
But there are differences in the activities of these proteins; these differences in activities are given in Table 11. With the proteins being the same in many ways (Table I), an explanation for the differences in biochemical and biological activities has to come from some other differences in properties. The difference in conformation seems to be related to these differences in activities. In direct support of this concept is the report by Frohman et a/. (1971) in which active tryptophan transport was found to be correlated with fH, with the correlation coefficient, r = 0.851, and P < 0.001. Some of the other differences in biochemical activities are of particular interest. One of these is the elevated lactate: pyruvate (L:P) ratio (Frohman et a/., 1960a,c); among several diseases this elevated L : P ratio was found only in schizophrenia (Frohman et a / . , 1962). The L:P ratio for schizophrenic patients also had large fluctuations, while the L : P ratio for control subjects was found to be steady (Frohman et a/., 1963). In the stressed schizophrenic patient, carbohydrate metabolism was largely through the hexose monophosphate shunt, instead of the Embden-Meyerhof pathway (Frohman er a/., 1960a,b). In the presence of the a-helical protein there was active tryptophan transport into rat brain cells (Warner et a/., 1972). The nonhelical protein lacked these activities. There are also two in cico differences in activities of these two forms of the protein. When treated with the protein
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from a patient’s plasma, rats that were trained to climb a rope for a food reward were unable to do so promptly (Bergen er al., 1962). Recently it was found that when electrodes were implanted into rat brains at the “pleasure center” (median forebrain bundle), and the rats were trained to press a bar to receive pleasurable stimuli, they did so incessantly. When the a-helical protein was injected intraventricularly into these rats, they slowed in pressing the bar; with the nonhelical protein, there was no rate change in pressing the bar (D. F. Caldwell et a/., unpublished data). There is some overlap reported in these conformational studies: some patients at times did not have the a-helical conformation, and nine of the control subjects a t times had small amounts of a-helical conformation. This latter situation may have occurred because these donors at the time of plasmapheresis were under great stress. For four of these control protein samples, one subject had been up all night (contrary to directions); another had undiagnosed pneumonia a t the time, and spent two weeks in bed after his plasmapheresis; and the other two had been subjected to other stresses. None of these four control subjects were included in this report. For the five
CONFORMATIONAL VARIATION
IN A
PLASMA
control samples which were included in this report, the plasmapheresis was a year or more in the past, and it was impossible to pinpoint lack of sleep, the presence of illness, etc. (These studies o n the effects of stress on the a-2 globulin conformation are being continued, but it has not been possible to plan ahead for such studies, in spite of efforts in this direction.) Other plasma parameters have been reported to vary with stress: L:P (Frohman et ul., 1966), plasma cholesterol (Wertlake et ul., 1958), phosphatidylglycerol (Austin, 1969), and plasma NEFA (Bogdonoff et a/., 1959) were all found to be elevated. The results of these conformation studies were examined by statistical analysis. By the chi-square test, P
