Prog. Neuro-Psychophormocol. 6 Biol. Psychiot. Printed in Great Britain. All rights reserved
1990. Vol. 14. pp. 553-561 0
0278-5846190 $0.00 + .5ll 1990 Pergamon Press plc
INFLUENCE OF PROTEIN CONCENTRATION ON PLATELET 3H-IMIPRAMINE BINDING EDWARD
M. DEMET, ALEKSANDRA
CHICZ-DEMET
and EVAN SHAFFER
Department of Psychiatry and Human Behavior, University of California, Irvine, CA USA
(Final form, February 1990)
DeMet, Edward M., Aleksandra Chin-DeMet and Evan Shaffer: Influence of Protein Concentrations crrlPlatelet ?I-Imipramine Binding. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1990,B: 5531. The effects of protein concentration in the assay mixture on platelet 3H-imipramine binding were studied in normal controls. 2. Increasing protein concentrations (76-926 ug/ml) were found to alter estimates of binding affinity (Kd) but not the number of binding sites (Bmax). 3. Increasing Kd estimates were protein dependent at concentrations in excess of ca. 200 ug/ml but were not protein dependent at lower concentrations. 4. A comparison of the present results with previous reports suggests that widespread interlaboratory differences in reported Bmax values for normal controls cannot be attributed to differences in the protein content of incubated samples. Rather these differences may be due to the inclusion of non-membrane protein in the assayed material. Keywords: depression, imipramine Abbreviations:
binding, membrane
protein, platelet membranes.
affinity binding constant (Kd), imipramine
(IMI), number of binding sites (Bmax).
Introductiog A number of studies now suggest that ‘H-imipramine
CH-IMI) binding may be lower in the brains
(Stanley et al. 1982) and platelets (Briley et al. 1980) of depressed patients than it is in normal controls.
However, reports of the actual number of binding sites (Bmax) and the affinity of binding
(Kd) vary greatly between laboratories
(Mellerup et al. 1982). While some of these differences may
be due to seasonal variations (Whitaker et al. 1984; DeMet et al. 1989), the widely discrepant values for normal controls clearly suggest the confounding influence of interlaboratory Such differences apparently
procedural differences.
exist in spite of the nearly universal use of similar binding conditions
(Briley et al. 1980). A notable exception is the quantity of protein used in the binding assays which varies widely between laboratories. that protein concentrations
Reported results from a number of laboratories
(Table 1) indicate
used in assay mixtures have varied from 116 ug/ml to over 3000 ug/ml.
553
E. M. DeMet et al.
554
In general, studies which have used lower protein concentrations
have tended to report higher Bmax
values and lower Kd values than studies which have used higher protein concentrations.
In this way,
Mellerup et al. (1982) have suggested that more reliable binding estimates may be obtained with lower protein concentrations.
Table 1 Platelet ‘H-Imipramine
Binding in Normal Controls
Bmax fmol/mg
Kd nM
Protein* ug/ml
,,is** Method
DeMet et al. 1989 Anderson et al. 1984
2647 1590
1.68 0.94
116 133-267
4 4
Theodorou Mellerup et etal.al.1982 1989 Marazziti et al. 1987 Meltzer and Arora 1986 Carstens et al. 1986 Nemeroff et al. 1988 Weizman et al. 1986 Raisman et al. 1981 Baron et al. 1983 Paul et al. 1980 Wood et al. 1983
1168 1010 1162 926 1300 908 517 581 991 625 754
0.64 0.96 1.78 0.91 1.20 0.94 1.53 2.40 3.40 1.40 2.57
167 150 120-273 240280 400 400 400-571 504-648 800 200-1200 2800-3600
% 2 3 2 3
Group
; 3 2 1
* Protein values are given per milliliter of incubated sample. ** Methods used for platelet lysis: (1) osmotic shock only; (2) osmotic shock with mechanical homogenization; (3) osmotic shock with polytron or ultra-turrax; (4) osmotic shock with sonication. While the potential importance (Bennett 1985; Hollenberg concentration
of protein concentration
in equilibrium
binding is well established
and Cuatrecasas 1979), few studies have examined the effects of protein
on platelet ‘H-IMI binding.
Two studies which utilized varying protein concentrations,
rather than fixed levels, have reported significant correlations between binding parameters and protein levels (Arora et al. 1985; Theodorou et al. 1989). Arora et al. (1985) found that Bmax was significantly inversely correlated with protein concentration relationship
in normal controls and depressed patients although this
was not significant when the depressed patients were separated into psychotic vs non-
psychotic subgroups.
The same study also found a significant positive correlation
levels and Kd values in psychotic depressed patients
although no correlation
between protein
was evident in the
depressed group as a whole or in normal controls. Theodorou et al. (1989) reported a slightly larger
Protein concentration and platelet “H-IMl binding
inverse relationship
555
between Bmax and protein levels such that Bmax values corrected for the effect
of protein concentration
were 1.4-18% higher than uncorrected values. In contrast, Innis et al. (1987)
failed to observe any effect of protein concentration
on platelet binding.
Only one study has directly addressed the tissue linearity of H-IMI binding with repeated measures of the same platelet pool (Barkai et al. 1985). The latter reported a robust linear increase in Kd values obtained using protein concentrations
in the range 100-800 ug protein but failed to find any
change in Bmax values. In this way one study has reported that Bmax and Kd are not dependent upon protein concentration, another found a Bmax dependence but no change in Kd, a third found a Kd dependence but no change in Bmax, and a fourth found modest correlations between protein and both Bmax and Kd. The present study re-examines the effects of varying protein concentration focus on lower protein values as employed by a multicenter
on Bmax and Kd estimates with a special study of the World Health Organization.
Methods
Whole blood samples were collected from normal volunteers between 0900 to 1000 h by platelet pheresis using a Fenwall model CS-3000 blood cell separator (Baxter-Fenwall,
Deerfield, IL). Normal
controls had no personal DSM-IIIR axis 1 or axis 2 disorders and no first degree relatives with an axis 1 disorder. Assessment Procedures Platelet isolation and W-IMI binding assays were performed by a modification
of the method of
Langer et al. (1980) as previously described (DeMet et al. 1989). Binding was evaluated at sixteen ‘HIMI concentrations ug/ml.
ranging from 0.2 to 5.5 nM and at six protein concentrations
ranging from 75-920
Binding constants (Bmax and Kd) were estimated by the method of Scatchard (1949) using
commercially
available software (program EBDA, Elsevier Biosoft, Cambridge,
proteins were determined using a commercially
UK).
Membrane
relative to bovine serum albumin (BSA) by the method of Bradford (1976)
prepared reagent (BioRad Laboratories,
Richmond,
CA).
Results The effects of membrane
protein on ‘H-IMI binding were evaluated at six concentrations
from 76 to 926 ug/rnl (final incubation
concentration).
values (expressed in molar concentration proportion
to membrane
ranging
The results in Fig 1 clearly show that Bmax
as output by the EBDA program)
protein over the range of concentrations
increased
in direct
commonly employed in binding
556
E. M. DcMet et al.
studies.
A Scatchard plot (not shown) at the lowest protein concentration
(r=0.980) over a range of ‘H-IMI concentrations
(76 ug/ml) was linear
such that the percentage of the total bound varied
between 3.7 to 16.5% (9 of 16 values at less than 10%). Similar Bmax estimates were obtained at this concentration
(Bmax= 188Ofmol/mg) and at the highest protein concentration
and the Hill coefficients were unaltered by the range of measurements the conditions of the assay at the lowest protein concentration the results indicate that Bmax values were essentially unaltered
0
200
400
600
(Bmax=2151 fmol/mg),
(range =0.945 to 0.999). Since
satisfy the criterion of Bennett (1985), by protein concentration
800
under the
1 30
MEMBRANE PROTEIN &/ml)
Fig 1. Effects of varying quantities of membrane
protein on the Bmax of platelet ‘H-IMI binding.
MEMBRANE PROTEIN (&ml)
Fig 2. Effects of protein concentration
on the Kd of platelet ‘H-IMI binding.
Protein
present conditions.
concentration
and platelet
557
“H-IMI binding
In contrast, Kd values (Fig 2) showed a marked inflection at ca. 200 q/ml
were increased by nearly three fold at the highest concentration
and
measured.
values The present study examined Bmax and Kd values obtained from a common platelet pool over a broad range of membrane
protein concentrations.
The number of ‘H-MI binding sites (Bmax) was
found to increase linearly with the amount of incubated protein over the entire range tested (76-926 ug/ml) and this relationship difference was obtained
could be extrapolated to a zero protein concentration.
at the lowest and highest protein concentrations
expressed in terms of milligrams protein.
In this way no
when these values were
The present results are in agreement with those obtained
by Barkai et al. (1985) and Innis et al. (1987) who also found no effect of protein on Bmax values, but differ from reports by Arora et al. (1985) and Theodorou et al. (1989) who found that Bmax values were protein dependent.
yof Eff
Kd V
On the other hand, Kd values were substantially
elevated at higher protein concentrations
nearly three fold difference between the highest and lowest protein concentrations
with a
tested. In this way
the present results replicate earlier findings by Barkai et al. (1985) who found a similar dependence at extreme differences in protein concentrations.
However, the present study examined a slightly larger
range than the earlier study and found that Kd values varied with protein concentration rather than a linear fashion.
in a sigmoidal
A finding of sigmoidal kinetics combined with an effect on Kd but not
Bmax strongly supports a hypothesis that these effects may be due to the accumulation endogenous competitive inhibitor at high membrane concentrations
of an
(Barkai et al. 1985). Several other
studies have suggested the possible presence of such an inhibitor (Barbaccia et al. 1984; Brusov et al. 1985). Nevertheless, the fact that Kd values were similar at protein concentrations
less than ca. 200
ug/ml suggests that valid Kd estimates may be made at these lower protein concentrations. Protein concentrations
may influence apparent binding parameters in different ways depending upon
the source of the measured protein.
Inclusion of excessive specific binding protein in an incubation
mixture would reduce the concentration
of free ligand thereby increasing
the apparent
Kd and
decreasing the apparent Bmax with similar effects on both measures. Just such a relationship is evident in a comparison of extreme values reported in Table 1. However, several studies which employed relatively large protein concentrations,
and found low Bmax values, have also reported relatively low
estimates of Kd. Paradoxically, the present results suggest that elevated protein levels should have
558
E. M. DeMet et al.
resulted in relatively high Kd values with little or no change in Bmax. Since this relationship
is not
evident in Table 1, the results suggest that variations in Kd and Bmax values reported in the literature are not solely attributable
of specific binding
protein.
Furthermore, the fact that Kd but not Bmax is altered by increasing membrane concentrations
suggests
that the concentration
proteins.
Protein on Bin-
proteins
Inclusion
concentration
concentrations
of free ligand is not significantly altered over a wide range of protein values.
Effects of Non-Bindine Non-binding
to the use of different
may alter binding estimates in a manner which is different from binding of such material
in the incubation
mix would not substantially
alter the
of free ligand. Therefore, excessive non-binding protein would decrease Bmax estimates
(expressed per mg protein) but would not alter estimates of Kd. In this way, a number of studies which have used relatively high protein concentrations
(A00
ug/ml), which might be expected to have
resulted in elevated Kd values (Barkai et al. 1985; present results), have instead reported relatively low estimates of both Bmax and Kd (Table 1). Non-binding
protein &tld be included in incubated samples as a result of incomplete platelet lysis.
Previous studies (Barber et al. 1971; Fried1 et al. 1983) have shown that conventional techniques
are not adequate
to insure complete platelet breakage.
homogenization
Similar studies in our own
laboratory (to be reported elsewhere) indicate that over 30 passes are required to break platelets with a Potter-Elvehjem
type homogenizer
under hypotonic conditions.
In contrast, ultrasonic treatment
results in complete cell breakage as verified by a lack of platelet counts in the present study. Various methods used for cell breakage by previous studies are summarizd
in Table 1. While this comparison
is imprecise, the results generally show that studies which have used more stringent methods for platelet breakage have tended to report higher Bmax values than studies which have used less stringent methods.
In addition
to non-specific
protein,
incomplete
platelet
lysis may also decrease
the
effectiveness of subsequent wash steps. In this regard it is notable that Anderson et al. (1984) found that Bmax values of frozen platelet membranes were increased by ca. two-fold by a subsequent wash step. Presumably this increase reflects the removal of either endogenous serotonin or an endogenous binding ligand (Barbaccia et al. 1984; Brusov et al. 1985) which may be facilitated by more disruptive techniques of cell breakage.
Conclusions The present results indicate that excessive specific binding protein concentrations
in incubated
samples can significantly increase Kd estimates above their actual values. However, these effects are largely restricted
to protein concentrations
apparently altered by concentrations
greater than ca. 200 ug/rnl and Bmax values are not
up to 900 ug/ml.
Therefore, excessive binding protein does nor
Protein
concentration
and platelet
559
“H-IMI binding
appear to explain widespread differences in Bmax estimates from normal controls.
Neither does it
explain patient-control
differences in platelet binding since most studies have reported similar Kd
values in these groups.
Rather it appears possible that an apparent dependence
protein concentrations
This research
of Bmax values on
may be due to the presence of unlysed cells in the incubation
was supported
by a grant from the National
Institute
of Mental
mixture.
Health
(ROl
MH40468). Referent ANDERSON, G.M., MINDERAA, R., VAN BENTHAM, (1984) Platelet Imipramine Binding in Autistic Subjects.
P., VOLKMAR, R. and COHEN, D.J. Psychiat. Res. 11: 133-141.
ARORA, R.C., WUNNICKE, V. and MELTZER, H.Y. (1985) Effect of Protein Concentration on Kinetic Constants (Kd and Bmax) of ‘H-Imipramine Binding in Blood Platelets. Biol. Psychiat. 2Q: 94-119. BARBACCIA, M.L, GANDOLFI, O., CHUANG, D.M. and COSTA, E. (1984) Autocoids for Drug Receptors: A New Approach in Drug Development. Proc. Natl. Acad. Sci. (USA) 8Q: 5134-5138. BARBER, Lysis and Brinkous, Stuttgart,
A.J., PEPPER, D.S. and JAMIESON, GA. (1971) A Comparison of Methods for Platelet the Isolation of Platelet Membranes. In: Thrombosis et. Diathesis Haemorrhagica, KM. E. Deutsch, R. Gross, J.E. Jorpes and F. Koller (Eds.), pp 38-57, Schattauer Verlag, New York.
BARKAI, AI., KOWALIK, S. and BARON, M. (1985) Effect of Membrane Protein Concentration on Binding of 3H-Imipramine in Human Platelets. Biol. Psychiat. a: 199-228. BARON, M., BARKAI, A., GRUEN, R., KOWALIK, S. and QUITKIN, F. (1983) 3H-Imipramine Platelet Binding Sites in Unipolar Depression. Biol. Psychiat. us: 1403-1409. BENNETT, J.P. (1985) Methods in Binding Studies. In: Neurotransmitter Receptor Binding, H.I. Yamamura, S.J. Enna and MJ. Kuhar (Eds.), pp 61-89, Raven Press, New York. BRADFORD, M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 2: 248-254. BRILEY, M.S., LANGER, S.Z., RAISMAN, R., SECHTER, D. and ZARIFIAN, E. (1980) Tritiated Imipramine Binding Sites are Decreased in Platelets of Untreated Depressed Patients. Science m: 303-305. BRUSOV, O.S., FOMENKO, A.M. and KATASONOV, AB. (1985) Human Plasma Inhibitors of Platelet Serotonin Uptake and Imipramine Receptor Binding: Extraction and Heterogeneity. Biol. Psychiat. a: 235-244. CARSTENS, M.E., ENGELBRECHT, AH., RUSSEL, VA., AALBERS, C., GAGIANO, C., CHALTON, D.O. and TALJAARD, J. (1986) Imipramine Binding Sites on Platelets of Patients with Major Depressive Disorder. Psychiat. Res. us: 333-342. DEMET, E.M., CHICZ-DEMET, A. and FLEISCHMANN, J. (1989) Seasonal Rhythm of Platelet “H-Imipramine Binding in Normal Controls. Biol. Psychiat. 26: 489-495.
560
E. M. DeMet
et al.
FRIEDL, W., PROPPING, P. and WECK, B. (1983) %I-Imipramine Binding in Platelets: Influence of Varying Proportions of Intact Platelets in Membrane Preparations on Binding. Psychopharmacol. j&j: 96199:
-
HOLLENBERG, M.D. and CUATRECASAS, P. (1979) Distinction of Receptor from Nonreceptor Interactions in Binding Studies. In: The Receptors. A Comprehensive Treatise, R.D. O’Brien, (Ed.), pp 193-214, Plenum Press, New York. INNIS, R.B., CHARNEY, D.S. and HENINGER, G.R. (1987) Differential 3H-Imipramine Binding in Patients with Panic Disorder and Depression. Psychiat. Res. 21: 33-41.
Platelet
LANGER, S.Z., BRILEY, M.S., RAISMAN, R., HENRY, J-F. and MORSELLI, P.L (1980) Specific 3H-Imipramine Binding in Human Platelets: Influence of Age and Sex. Naunyn Schmiedebergs Arch. Pharmacol. m: 189-194. MARAZZITI, D., GIUSTI, P., ROTONDO, A, PLACIDI, G.F. and PACIFICI, G.M. (1987) Imipramine Receptors in Human Platelets: Effects of Age. Int. J. Clin. Pharmcol. Res. m: 145 148. MELLERUP, E.T., PLENGE, P. and ROSEBERG, R. (1982) Platelets from Psychiatric Patients. Psychiat. Res. 1: 221-227.
3H-Imipramine
Binding Sites in
MELTZER, H.Y. and ARORA, R.C. (1986) Platelet Markers of Suicidal@. In: Psychobiology of Suicidal Behavior, J.J. Mann and M. Stanley (Eds.), pp 271-280, Ann. N.Y. Acad. Sci. QZ, The New York Academy of Sciences, New York. NEMEROFF, C.B., KNIGHT, D.L., KRISHNAN, R.R., SLOTKIN, T.A., BISSE’ITE, G., MELVILLE, M. and BLAZER, D.G. (1988) Marked Reduction in the Number of Platelet-Tritiated Imipramine Binding Sites in Geriatric Depression. Arch. Gen. Psychiat. &$ 919-923. PAUL, S.M., REHAVI, M., SKOLNICK, P. and GOODWIN, F.K (1980) Demonstration of Specific “High Affinity” Binding Sites for 3H-Imipramine on Human Platelets. Life Sci. 26: 953-959. RAISMAN, R., SECHTER, D., BRILEY, M.S., ZARIFIAN, E. and LANGER, S.Z. (1981) HighAffinity ‘H-Imipramine Binding in Platelets from Untreated and Treated Depressed Patients Compared to Healthy Volunteers. Psychopharmacol. 25: 368-371. SCATCHARD, G. (1949) The Attractions Acad. Sci. a: 660-672.
of Proteins for Small Molecules and Ions. Ann. N.Y.
STANLEY, M., VIRGILIO, J. and GERSHON, S. (1982) Tritiated Imipramine Decreased in the Frontal Cortex of Suicides. Science m: 1337-1339.
Binding Sites are
THEODOROU, GE., KATONA, C.L., DAVIES, S.L., HALE, AS., KERRY, S.M., HORTON, R.W., KELLEY, J.S. and PAYKEL, E.S. (1989) ‘H-Imipramine Binding to Freshly Prepared Platelet Membranes in Depression. Psychiat. Res. 29: 87-103. WEIZMAN, R., CARMI, M., TYANO, S. and REHAVI, M. (1986) Reduced 3H-Imipramine Binding but Unaltered ‘H-Serotonin Uptake in Platelets of Adolescent Enuretics. Psychiat. Res. B: 37-42. WHITAKER, P.M., WARSH, J.J., STANCER, H.C., PERSAD, E. and VINT, C.K. (1984) Seasonal Variations in Platelet “H-Imipramine Binding: Comparable Values in Control and Depressed Populations. Psychiat. Res. 11: 127-131. WOOD, P., CADOTTE, B., NAIR, N., LAFAILLE, F. and SCHWARTZ, G. (1983) Lack of Association between “H-Imipramine Binding Sites and Uptake of Serotonin in Control, Depressed and Schizophrenic Patients. Neuropharmacol. 22: 1211-1214.
Protein
concentration
and platelet
Inquiries and reprint requests should be addressed to:
Dr. Edward M. DeMet University of California, Irvine Department of Psychiatry and Human Behavior, Medical Sciences Bldg. I, Rm. D435, Irvine, CA 92717 U.S.A.
“H-IMI binding
561
1990. Vol. 14. pp. 553-561 0
0278-5846190 $0.00 + .5ll 1990 Pergamon Press plc
INFLUENCE OF PROTEIN CONCENTRATION ON PLATELET 3H-IMIPRAMINE BINDING EDWARD
M. DEMET, ALEKSANDRA
CHICZ-DEMET
and EVAN SHAFFER
Department of Psychiatry and Human Behavior, University of California, Irvine, CA USA
(Final form, February 1990)
DeMet, Edward M., Aleksandra Chin-DeMet and Evan Shaffer: Influence of Protein Concentrations crrlPlatelet ?I-Imipramine Binding. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1990,B: 5531. The effects of protein concentration in the assay mixture on platelet 3H-imipramine binding were studied in normal controls. 2. Increasing protein concentrations (76-926 ug/ml) were found to alter estimates of binding affinity (Kd) but not the number of binding sites (Bmax). 3. Increasing Kd estimates were protein dependent at concentrations in excess of ca. 200 ug/ml but were not protein dependent at lower concentrations. 4. A comparison of the present results with previous reports suggests that widespread interlaboratory differences in reported Bmax values for normal controls cannot be attributed to differences in the protein content of incubated samples. Rather these differences may be due to the inclusion of non-membrane protein in the assayed material. Keywords: depression, imipramine Abbreviations:
binding, membrane
protein, platelet membranes.
affinity binding constant (Kd), imipramine
(IMI), number of binding sites (Bmax).
Introductiog A number of studies now suggest that ‘H-imipramine
CH-IMI) binding may be lower in the brains
(Stanley et al. 1982) and platelets (Briley et al. 1980) of depressed patients than it is in normal controls.
However, reports of the actual number of binding sites (Bmax) and the affinity of binding
(Kd) vary greatly between laboratories
(Mellerup et al. 1982). While some of these differences may
be due to seasonal variations (Whitaker et al. 1984; DeMet et al. 1989), the widely discrepant values for normal controls clearly suggest the confounding influence of interlaboratory Such differences apparently
procedural differences.
exist in spite of the nearly universal use of similar binding conditions
(Briley et al. 1980). A notable exception is the quantity of protein used in the binding assays which varies widely between laboratories. that protein concentrations
Reported results from a number of laboratories
(Table 1) indicate
used in assay mixtures have varied from 116 ug/ml to over 3000 ug/ml.
553
E. M. DeMet et al.
554
In general, studies which have used lower protein concentrations
have tended to report higher Bmax
values and lower Kd values than studies which have used higher protein concentrations.
In this way,
Mellerup et al. (1982) have suggested that more reliable binding estimates may be obtained with lower protein concentrations.
Table 1 Platelet ‘H-Imipramine
Binding in Normal Controls
Bmax fmol/mg
Kd nM
Protein* ug/ml
,,is** Method
DeMet et al. 1989 Anderson et al. 1984
2647 1590
1.68 0.94
116 133-267
4 4
Theodorou Mellerup et etal.al.1982 1989 Marazziti et al. 1987 Meltzer and Arora 1986 Carstens et al. 1986 Nemeroff et al. 1988 Weizman et al. 1986 Raisman et al. 1981 Baron et al. 1983 Paul et al. 1980 Wood et al. 1983
1168 1010 1162 926 1300 908 517 581 991 625 754
0.64 0.96 1.78 0.91 1.20 0.94 1.53 2.40 3.40 1.40 2.57
167 150 120-273 240280 400 400 400-571 504-648 800 200-1200 2800-3600
% 2 3 2 3
Group
; 3 2 1
* Protein values are given per milliliter of incubated sample. ** Methods used for platelet lysis: (1) osmotic shock only; (2) osmotic shock with mechanical homogenization; (3) osmotic shock with polytron or ultra-turrax; (4) osmotic shock with sonication. While the potential importance (Bennett 1985; Hollenberg concentration
of protein concentration
in equilibrium
binding is well established
and Cuatrecasas 1979), few studies have examined the effects of protein
on platelet ‘H-IMI binding.
Two studies which utilized varying protein concentrations,
rather than fixed levels, have reported significant correlations between binding parameters and protein levels (Arora et al. 1985; Theodorou et al. 1989). Arora et al. (1985) found that Bmax was significantly inversely correlated with protein concentration relationship
in normal controls and depressed patients although this
was not significant when the depressed patients were separated into psychotic vs non-
psychotic subgroups.
The same study also found a significant positive correlation
levels and Kd values in psychotic depressed patients
although no correlation
between protein
was evident in the
depressed group as a whole or in normal controls. Theodorou et al. (1989) reported a slightly larger
Protein concentration and platelet “H-IMl binding
inverse relationship
555
between Bmax and protein levels such that Bmax values corrected for the effect
of protein concentration
were 1.4-18% higher than uncorrected values. In contrast, Innis et al. (1987)
failed to observe any effect of protein concentration
on platelet binding.
Only one study has directly addressed the tissue linearity of H-IMI binding with repeated measures of the same platelet pool (Barkai et al. 1985). The latter reported a robust linear increase in Kd values obtained using protein concentrations
in the range 100-800 ug protein but failed to find any
change in Bmax values. In this way one study has reported that Bmax and Kd are not dependent upon protein concentration, another found a Bmax dependence but no change in Kd, a third found a Kd dependence but no change in Bmax, and a fourth found modest correlations between protein and both Bmax and Kd. The present study re-examines the effects of varying protein concentration focus on lower protein values as employed by a multicenter
on Bmax and Kd estimates with a special study of the World Health Organization.
Methods
Whole blood samples were collected from normal volunteers between 0900 to 1000 h by platelet pheresis using a Fenwall model CS-3000 blood cell separator (Baxter-Fenwall,
Deerfield, IL). Normal
controls had no personal DSM-IIIR axis 1 or axis 2 disorders and no first degree relatives with an axis 1 disorder. Assessment Procedures Platelet isolation and W-IMI binding assays were performed by a modification
of the method of
Langer et al. (1980) as previously described (DeMet et al. 1989). Binding was evaluated at sixteen ‘HIMI concentrations ug/ml.
ranging from 0.2 to 5.5 nM and at six protein concentrations
ranging from 75-920
Binding constants (Bmax and Kd) were estimated by the method of Scatchard (1949) using
commercially
available software (program EBDA, Elsevier Biosoft, Cambridge,
proteins were determined using a commercially
UK).
Membrane
relative to bovine serum albumin (BSA) by the method of Bradford (1976)
prepared reagent (BioRad Laboratories,
Richmond,
CA).
Results The effects of membrane
protein on ‘H-IMI binding were evaluated at six concentrations
from 76 to 926 ug/rnl (final incubation
concentration).
values (expressed in molar concentration proportion
to membrane
ranging
The results in Fig 1 clearly show that Bmax
as output by the EBDA program)
protein over the range of concentrations
increased
in direct
commonly employed in binding
556
E. M. DcMet et al.
studies.
A Scatchard plot (not shown) at the lowest protein concentration
(r=0.980) over a range of ‘H-IMI concentrations
(76 ug/ml) was linear
such that the percentage of the total bound varied
between 3.7 to 16.5% (9 of 16 values at less than 10%). Similar Bmax estimates were obtained at this concentration
(Bmax= 188Ofmol/mg) and at the highest protein concentration
and the Hill coefficients were unaltered by the range of measurements the conditions of the assay at the lowest protein concentration the results indicate that Bmax values were essentially unaltered
0
200
400
600
(Bmax=2151 fmol/mg),
(range =0.945 to 0.999). Since
satisfy the criterion of Bennett (1985), by protein concentration
800
under the
1 30
MEMBRANE PROTEIN &/ml)
Fig 1. Effects of varying quantities of membrane
protein on the Bmax of platelet ‘H-IMI binding.
MEMBRANE PROTEIN (&ml)
Fig 2. Effects of protein concentration
on the Kd of platelet ‘H-IMI binding.
Protein
present conditions.
concentration
and platelet
557
“H-IMI binding
In contrast, Kd values (Fig 2) showed a marked inflection at ca. 200 q/ml
were increased by nearly three fold at the highest concentration
and
measured.
values The present study examined Bmax and Kd values obtained from a common platelet pool over a broad range of membrane
protein concentrations.
The number of ‘H-MI binding sites (Bmax) was
found to increase linearly with the amount of incubated protein over the entire range tested (76-926 ug/ml) and this relationship difference was obtained
could be extrapolated to a zero protein concentration.
at the lowest and highest protein concentrations
expressed in terms of milligrams protein.
In this way no
when these values were
The present results are in agreement with those obtained
by Barkai et al. (1985) and Innis et al. (1987) who also found no effect of protein on Bmax values, but differ from reports by Arora et al. (1985) and Theodorou et al. (1989) who found that Bmax values were protein dependent.
yof Eff
Kd V
On the other hand, Kd values were substantially
elevated at higher protein concentrations
nearly three fold difference between the highest and lowest protein concentrations
with a
tested. In this way
the present results replicate earlier findings by Barkai et al. (1985) who found a similar dependence at extreme differences in protein concentrations.
However, the present study examined a slightly larger
range than the earlier study and found that Kd values varied with protein concentration rather than a linear fashion.
in a sigmoidal
A finding of sigmoidal kinetics combined with an effect on Kd but not
Bmax strongly supports a hypothesis that these effects may be due to the accumulation endogenous competitive inhibitor at high membrane concentrations
of an
(Barkai et al. 1985). Several other
studies have suggested the possible presence of such an inhibitor (Barbaccia et al. 1984; Brusov et al. 1985). Nevertheless, the fact that Kd values were similar at protein concentrations
less than ca. 200
ug/ml suggests that valid Kd estimates may be made at these lower protein concentrations. Protein concentrations
may influence apparent binding parameters in different ways depending upon
the source of the measured protein.
Inclusion of excessive specific binding protein in an incubation
mixture would reduce the concentration
of free ligand thereby increasing
the apparent
Kd and
decreasing the apparent Bmax with similar effects on both measures. Just such a relationship is evident in a comparison of extreme values reported in Table 1. However, several studies which employed relatively large protein concentrations,
and found low Bmax values, have also reported relatively low
estimates of Kd. Paradoxically, the present results suggest that elevated protein levels should have
558
E. M. DeMet et al.
resulted in relatively high Kd values with little or no change in Bmax. Since this relationship
is not
evident in Table 1, the results suggest that variations in Kd and Bmax values reported in the literature are not solely attributable
of specific binding
protein.
Furthermore, the fact that Kd but not Bmax is altered by increasing membrane concentrations
suggests
that the concentration
proteins.
Protein on Bin-
proteins
Inclusion
concentration
concentrations
of free ligand is not significantly altered over a wide range of protein values.
Effects of Non-Bindine Non-binding
to the use of different
may alter binding estimates in a manner which is different from binding of such material
in the incubation
mix would not substantially
alter the
of free ligand. Therefore, excessive non-binding protein would decrease Bmax estimates
(expressed per mg protein) but would not alter estimates of Kd. In this way, a number of studies which have used relatively high protein concentrations
(A00
ug/ml), which might be expected to have
resulted in elevated Kd values (Barkai et al. 1985; present results), have instead reported relatively low estimates of both Bmax and Kd (Table 1). Non-binding
protein &tld be included in incubated samples as a result of incomplete platelet lysis.
Previous studies (Barber et al. 1971; Fried1 et al. 1983) have shown that conventional techniques
are not adequate
to insure complete platelet breakage.
homogenization
Similar studies in our own
laboratory (to be reported elsewhere) indicate that over 30 passes are required to break platelets with a Potter-Elvehjem
type homogenizer
under hypotonic conditions.
In contrast, ultrasonic treatment
results in complete cell breakage as verified by a lack of platelet counts in the present study. Various methods used for cell breakage by previous studies are summarizd
in Table 1. While this comparison
is imprecise, the results generally show that studies which have used more stringent methods for platelet breakage have tended to report higher Bmax values than studies which have used less stringent methods.
In addition
to non-specific
protein,
incomplete
platelet
lysis may also decrease
the
effectiveness of subsequent wash steps. In this regard it is notable that Anderson et al. (1984) found that Bmax values of frozen platelet membranes were increased by ca. two-fold by a subsequent wash step. Presumably this increase reflects the removal of either endogenous serotonin or an endogenous binding ligand (Barbaccia et al. 1984; Brusov et al. 1985) which may be facilitated by more disruptive techniques of cell breakage.
Conclusions The present results indicate that excessive specific binding protein concentrations
in incubated
samples can significantly increase Kd estimates above their actual values. However, these effects are largely restricted
to protein concentrations
apparently altered by concentrations
greater than ca. 200 ug/rnl and Bmax values are not
up to 900 ug/ml.
Therefore, excessive binding protein does nor
Protein
concentration
and platelet
559
“H-IMI binding
appear to explain widespread differences in Bmax estimates from normal controls.
Neither does it
explain patient-control
differences in platelet binding since most studies have reported similar Kd
values in these groups.
Rather it appears possible that an apparent dependence
protein concentrations
This research
of Bmax values on
may be due to the presence of unlysed cells in the incubation
was supported
by a grant from the National
Institute
of Mental
mixture.
Health
(ROl
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Platelet
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3H-Imipramine
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Binding Sites are
THEODOROU, GE., KATONA, C.L., DAVIES, S.L., HALE, AS., KERRY, S.M., HORTON, R.W., KELLEY, J.S. and PAYKEL, E.S. (1989) ‘H-Imipramine Binding to Freshly Prepared Platelet Membranes in Depression. Psychiat. Res. 29: 87-103. WEIZMAN, R., CARMI, M., TYANO, S. and REHAVI, M. (1986) Reduced 3H-Imipramine Binding but Unaltered ‘H-Serotonin Uptake in Platelets of Adolescent Enuretics. Psychiat. Res. B: 37-42. WHITAKER, P.M., WARSH, J.J., STANCER, H.C., PERSAD, E. and VINT, C.K. (1984) Seasonal Variations in Platelet “H-Imipramine Binding: Comparable Values in Control and Depressed Populations. Psychiat. Res. 11: 127-131. WOOD, P., CADOTTE, B., NAIR, N., LAFAILLE, F. and SCHWARTZ, G. (1983) Lack of Association between “H-Imipramine Binding Sites and Uptake of Serotonin in Control, Depressed and Schizophrenic Patients. Neuropharmacol. 22: 1211-1214.
Protein
concentration
and platelet
Inquiries and reprint requests should be addressed to:
Dr. Edward M. DeMet University of California, Irvine Department of Psychiatry and Human Behavior, Medical Sciences Bldg. I, Rm. D435, Irvine, CA 92717 U.S.A.
“H-IMI binding
561
