190, No. 1, September,





pp. 109-117,


Cyclase in Polyacrylamide Gel Electrophoresis: but Active C. NEWBY,*’


* Section on Membrane Regulation, Arthritis, Metabolism and Digsestive National Institute of Child Health Received






Laboratory of Nutrition and Endocrinology, National Institute of Diseases, and t Endocrinology and Reproduction Research Branch, and Human Development, National Institutes of Health, Bethesda, Maryland 20014 December

16, 1977; revised



An intrinsic “insoluble” membrane-bound enzyme, adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC was analyzed in a detergent solubilized but active form by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis provided improved resolution of the enzyme from contaminant proteins compared to previously used column chromatographic procedures, and allowed the analysis of much smaller quantities of material. The success of this approach depended on a particular choice of the nature and concentration of the detergent, of pH, of buffer constituents, of temperature, and of gel architecture. Using either the basal enzyme or enzyme pretreated in the membrane with guanyl-5’-yl imidodiphosphate (Gpp(NH)p) in the presence or absence of glucagon, up to 90% of enzymatic activity was recovered. However, the basal enzyme lost sensitivity to stimulation by Gpp(NH)p but not fluoride during electrophoresis. Ferguson plot analysis was used to determine the size and charge parameters of the enzyme, and bandwidth to estimate the degree of polydispersity. Increasing detergent concentrations reduced the effective size and degree of polydispersity of the enzyme, although this also led to progressive inactivation. The size of the enzyme was independent of preactivation by Gpp(NH)p alone or Gpp(NH)p plus glucagon. The “smallest and least polydisperse” size to which the active enzyme could be reduced was 3.6 f 0.8 to 4.0 f 0.8 nm (geometric mean radius).

Polyacrylamide gel electrophoresis is, at present, the one fractionation method capable of simultaneously exploiting differences among macromolecules in all of their major properties: charge, size and relative hydrophobicity (1). If a specific assay is available, it also lends itself to the characterization of active macromolecules in a crude mixture. PAGE2 in strongly ionic detergents, while overcoming the insolubility of membrane proteins, sacrifices most of

these advantages. The secondary, tertiary, and quaternary structure of the protein is disrupted, charges among molecules are equalized and activity is lost. Nonionic detergents can also be used to solubilize membrane proteins, but to a lesser and as yet ill-defined extent. In order to be useful for characterization or purification, a condition of solubilization must be achieved which disrupts interactions between different proteins while preserving active (glyco)protein-lipid-detergent aggregates. This report describes an attempt to characterize the structure of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC produced by this kind of specific detergent dispersion of the rat liver plasma membrane. The approach necessitated the design of a PAGE fractionation system in which adequate electrophoretic mobility could be achieved without sacrificing enzymatic activity.

’ Present address: University of Cambridge, Department of Clinical Biochemistry, Addenbrooke’s Hospital, Hills Rd., Cambridge CB2 ZQR, Great Britam. * Abbreviations used: Gpp(NH)p, guanyl-5’-yl imidodiphosphate, PAGE, polyacrylamide gel electrophoresis; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid; Kx, retardation coefficient; Yo, free electrophoretic mobility parameter; EPPS, N-(2hydroxyethylj-1-piperazinepropanesulfonic acid. 109

0003-9861/78/1901-0109$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.






The following sources of materials were used: Thyroglobulin, ceruloplasmin, P-galactosidase (P-n-galactoside galactohydrolase, EC, /I-lactoglobulin, creatine phosphokinase (ATP:creatine phosphotransferase, EC and 4-(L-hydroxyethyl)-l-piperazinepropanesulphonic acid from Sigma; bovine serum albumin from Reheis Chemical Corp.; ferritin and ultrapure sucrose from Schwarz Mann; catalase (H20:H202 oxidoreductase, EC from Worthington; transferrin from Behring Diagnostics Inc.; [#P]ATP (lo-25 Ci/mmol), adenyl-5’-yl imidodiphosphate and guanyl-5’-yl imidodiphosphate from ICN; [G-“HIcAMP (30-50 Ci/mmole) from New England Nuclear; N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid from Calbiochem. Lubrol PX was a gift of ICI America Inc. Membranes and soluble fractions. Partially purified rat liver plasma membranes were prepared as described previously (2) and stored in liquid nitrogen before use. Adenylate cyclase was solubilized with Lubrol PX in a guanyl nucleotide sensitive form from hepatic membranes as described previously (3,4). The soluble enzyme was stable for several days at 4°C provided that buffers contained 25% (w/v) sucrose and 1 mM dithiothreitol (4). Higher activity states were produced by treatment, 37°C 10 min, of liver membranes with Gpp(NH)p, 0.01 mru, or Gpp(NH)p, 0.01 mru, plus glucagon, 2.10-’ M (3). Activated states were persistent after solubilization (3). Electrophoresis. The apparatus, procedures of polymerization and of gel electrophoresis used in this work have been described in detail (1). The solutions used were as follows. 1. Stacking gel polymerization mixture: 2.5 g of acrylamide, 0.625 g of NJ’-methylenebisacrylamide, 12.5 mg of potassium persulfate, 0.5 mg of riboflavin, 25 g of sucrose, 0.375 g of imidazole/HCl, per 100 ml, pH 6.0. Resolving gel polymerization mixture: 3.4 to 6.4 g of a&amide, 0.6 to 1.1 g of N,N’-diallyltartardiamide, 12.5 mg of potassium persulphate, 0.5 mg of riboflavin, 25 g of sucrose, 1.18 g of imidazole/HCl, per 100 ml, pH 7.5 (or 0.375 g of imidazole/HCl per 100 ml, pH 6.0). 2. Upper buffer, 10 times concentrated: 25.2 g of EPPS, 8.3 g of imidazole, 0.0744 g of EDTA.ZHzO per liter, pH 7.5. 3. Lower buffer: 0.05 M HCl and 0.0625 M imidazole, pH 5.8. Stacking gel (0.3 ml per gel) and resolving gel (1.6 ml per gel) polymerization mixtures were degassed at 10 mm Hg (1) for 5 min. N,NJV’,N’-Tetramethylethylenediamine (1 pi/ml) and Lubrol PX [to a final concentration of 0.1% (w/v)] were then added. After overlayering with water, polymerization was initiated at 0°C with fluorescent light (1,. Electrophoresis was conducted at 0°C at a constant current of 1.5 mA per cylindrical gel of 6 mm diameter. Soluble fraction (50 fl) (approximately 100 pg of



protein) containing 10 pi/ml of RBY (Gehnan no. 51250) tracking dye mixture was used. The red component was used to mark the front. Standard proteins were suspended to 1 mg/ml in upper buffer, dialyzed overnight vs. 1000 vol of upper buffer and then diluted 1:l (v/v) with upper buffer containing 50% sucrose. After addition of RBY dye they were subjected to PAGE as described. Gels were sectioned into 2-mm slices (Bio-Rad slicer no. 190), slices were suspended in assay cocktail and analyzed for activity. Alternatively, gels were stained for protein with Coomassie brilliant blue G-250 (5). Densitometry on stained protein zones was carried out by Gilford spectrophotometer (linear transport accessory) at 600 nm. Bandwidth at half-height was determined in R, units from the densitometry tracings and the hand-drawn activity profdes. Rf was determined for adenylate cyclase by dividing the slice number of peak activity by the slice number of the front moving boundary marked by tracking dye, and for standards using a digital electronic Rrmeasuring device (1). Ferguson plots were constructed from R/values determined at six gel concentrations or more. These plots were obtained by computerized linear regression analysis (6) which yielded values for the slope (KR, retardation coefficient, a measure of molecular size) and the antilog of the intercept on the log RI axis ( Yo, a measure of free electrophoretic mobility) and their confidence limits.. Values of KR were translated to those of geometric mean radius (R) by linear interpolation from a standard curve as described (6). Assays. Adenylate cyclase activity was determined at 30°C for 10 min as described previously (7). Gel slices were allowed to equilibrate with assay cocktail (100 ~1) for 2 h at O’C before assay. After termination (7), samples were left at 4°C overnight to extract the [3ZP]cAMP produced. GTPase was assayed by a similar modification of the previously described technique (4). Protein was determined according to Lowry et al.


Optimization of pH and Buffer Constituents for PAGE Initial attempts at PAGE using the Ornstein-Davis procedure (9) led to very low recoveries of adenylate cyclase activity. Since the operative pH of this buffer system is 10.2, O”C, inactivation may be explained by the pH-stability profile of the enzyme.3 Both the basal enzyme and the state induced by Gpp(NH)p plus glucagon displayed maximal stability over the range pH 6.5 to pH 9.0 and were inactivated by 50% after incubation for 3 h, 0°C at either pH 5.5 or pH 10.0. Thus a buffer system with 3 Supplementary quest.



on res





lower operative pH was sought. An empirical buffer system using EPPS, pK, 8.0, as trailing ion with stacking at pH 7.5 and a number of buffer systems* defined physicochemically by Jovin et al. (10, 11) were evaluated. Soluble fractions from pretreated and untreated membranes were electrophoresed into 1.5 ml stacking gels, varying the gel concentration, pH, stacking limits (1) and buffer systems until all the adenylate cyclase activity and stained protein that entered the gel was found in a sharp boundary at the dye front. Recovery of activity from the gel slice of the stack was optimized at this stage, with the following results. Conducting electrophoresis at 0°C was essential. Inclusion of 25% (w/v) sucrose improved recoveries several fold. (The same was later found for resolving gels.) Inclusion of Lubrol in the gel buffer was not necessary for maximal activity and pre-electrophoresis with thioglycolate (1)) to remove oxidized products of the polymerization reaction, did not improve recovery. Stacking was obtained with Jovin system number 2492.1 as well as with the empirical system. However, maximal recovery of enzymatic activity in system 2492.1 was 1576, whereas the empirical system achieved 5090% recovery irrespective of activity state. The empirical system was, therefore, selected. Ebctrophoretic


Multiphasic gel electrophoresis of soluble fractions from untreated and pretreated membranes led to similar electrophoretic profiles both for protein (Fig. 1) and adenylate cyclase activity. The enzyme migrated as a single major component with a small proportion (5-10s) failing to enter even the stacking gel (Fig. 2). The position of the peaks of adenylate cyclase activity 4 The evaluated:



Jovin system (10,ll) number 1944.1 2061.2 2514.1 2492.1 2492.1

+ 0.1






Results No stacking No stacking No stacking Stacking; low recovery enzymatic activity Stacking; no recovered activity





FIG. 1. Soluble fractions (50 ~1) from untreated rat liver plasma membranes were subjected to electrophoresis with 5.0% (w/v) total monomer and 0.1% Lubrol PX in the resolving gel. Gels were stained for protein according to Diezel et al. (5). The left-hand gel was obtained with an operative pH of 8.3 in the resolving gel. The right-hand gel was obtained when upper gel buffer was used in both stacking and resolving gels, in which case the operative pH was 7.5. The position of the dye front is indicated by F. The Rf of adenylate cyclase (C); the larger GTPase (G,); and the smaller GTPase (Gz); determined in parallel gels is indicated.

are indicated in Fig. 1 although in view of the highly impure preparations used none of the visible protein bands may correspond to the enzyme. Electrophoresis was initially conducted with Lubrol concentrations identical to those used previously in gel filtration experiments (3, 4). In that work, specific GTPases which co-chromatographed with the cyclase were described. Instead of a









FIG. 2. Electrophoresis of soluhilized adenylate cyclase pretreated with Gpp(NH)p plus glucagon as described was conducted at pH 8.3 at a total gel monomer concentration of 5% (w/v) in the resolving gel. Different concentrations of Lubrol PX were present in the stacking and resolving gels. (A) no Lubrol; m) 0.01% (w/v) Lubrol; (a) 0.1% (w/v) Lubrol. Adenylate cyclase was assayed with 50 PM ATP as substrate. Total recoveries of enzyme activity was 80%, 66% and 54%, respectively. No activity was detected in slices 16 to 27. 7


-10 -9 -8 -7

0 3D o_Q gz

-6 -5

$2 p;;l


;2 0


1 2

I 4

I 6

L I1 lb 1 I I I1 8 10 12 14 16 18 20 22 24 SLICE



1,. 26 28


FIG. 3. Electrophoretic profiles determined in replicate gels for adenylate cyclase and GTPase (assayed with 5 mM Mg”). Membranes were pretreated with Gpp(NH)p and glucagon, as described. A gel concentration of 6.0% and a pH of 7.5 in the resolving gel were used. Lubrol PX was present in stacking and resolving gels at 0.01% (w/v).

broad GTPase profile showing different metal ion preference at the leading and trailing edges, two components of activity were clearly resolved in PAGE (Fig. 3). The leading and trailing components showed the same metal ion dependencies as those seen in gel filtration (A. F. Welton and A. C. Newby, unpublished data). Both major peaks of GTPase activity were resolved from the cyclase5 although a minor portion ‘When analysis was carried out at six gel concentrations and Ferguson plots constructed, the three enzymes were significantly distinguishable by the cri-

did cochromatograph with the enzyme. These studies illustrated the superior resolving power of the PAGE system relative to the previous method. Loss of Gpp(NH)p Sensitivity of the Basal Enzyme Substantial loss of Gpp(NH)p-stimulation of basal cyclase activity occurred subsequent to or during electrophoresis, partial loss occurred in the stacking phase (Table terion of the nonoverlap of the joint 95% confidence envelopes of KR and Yo (6).





I). By contrast, the enzyme retained partial sensitivity to activation by fluoride throughout the electrophoresis. Although further studies wilI be required to delineate the basis of the loss of Gpp(NH)p-sensitivity, it is clear that the basal enzyme has been modified during electrophoresis (see Discussion). Bandwidth The Rf and bandwidth of the peaks of enzymatic activity obtained with each activity state of the cyclase was investigated as a function of Lubrol concentration. Increasing the concentration of Lubrol PX in the stacking and resolving gels led to a progressive narrowing of bandwidth (Fig. 2) but also to a loss of recovered activity. At this gel concentration, a small increase in Rf was also observed. Minimal bandwidths were observed at Lubrol concentrations of 0.1% (w/v) and above. Increasing Lubrol concentration to 1% (w/v) led to no further change in either Rf or bandwidth, although recovery of activity decreased to approximately 40%. These bandwidths were compared to those obtained by densitometry for the standard proteins listed in the legend to Fig. 7 by a previously described procedure (Fig. 2 of Ref. 7). It was seen (Fig. 4) that the bandwidth for cyclase activity at both 5.5 and 6.5% gel concentration lay outside the range of values obTABLE Gpp(NH)p Expt.



Electrophoretic St&IS



Basal Stacking resolution





tained for stained protein standards. Assuming that the sensitivities of the enzyme assay and of staining are similar, this indicated a higher degree of polydispersity for the cyclase than for the standards. Since stacking involves a considerable concentration of the proteins (l), it was important to test whether stacking itself caused any structural change, such as aggregation of the enzyme. Samples of soluble fractions were electrophoresed into resolving gels at several gel concentrations with and without stacking gels. No alterations in Rf were observed although bandwidths were increased in the absence of stacking. Ferguson Plot Analyses Performing electrophoresis at a series of gel concentrations allows one to determine both size and charge parameters of a protein (1). As shown by Ferguson (12), a plot of log Rf vs. gel concentration should be linear. Clearly the extrapolated value of Rf at zero gel concentration ( Yo) represents the electrophoretic mobility of the protein in free solution relative to the moving boundary and hence is related to net charge. The slope of the plot (KR) is a measure of the molecular sieving effect of the polyacrylamide gel and hence is related to molecular radius (6). Owing to the large size of the enzyme (see below), it was necessary to use highly I OF ADENYLATE

activitv I- pmoI/lO min + lo-5M GDPNHP






-Fold stimulation by activator

-Fold stimulation of original sample

Recoverv of basal a”& tivity (o/o)









2.42 2.57 7.60 7.20 0.76b 0.48’

2.28 3.21 1.36 1.00

29.3 22.7

0.94 1.2 3.8 3.2 1.8 2.1

4.2 4.5 9.6 9.6 4.5 4.5

31 54 93 88 226 16’

p Soluble fractions (50 ~1) from untreated liver plasma membranes were electrophoresed for 2 h into either stacking and resolving gels or (1.5 ml) stacking gels alone. Replicate gels were sliced and assayed either with or without activator at the level shown. Total recovered activity was computed by summing activities recovered from each slice of the gel. * The low recoveries in the case of the stacking step alone are thought to be due to the extended period that the enzyme remained stacked relative to the multiphasic gel experiments.


NEWBY, 5.5%



6.5 I% fw/vl TOTAL MONOMER




0 .






Rf h FIG. 4. Bandwidth at one-half peak height of standard proteins (by densitometry) and adenylate cyclase (by enzyme assay) was measured [(O) PAGE in 0.01% or (0) 0.1% Lubrol] at two gel concentrations (6). The standard proteins (0) were: At 5.5% (w/v) total monomer, ferritin, thyroglobulin, catalase, &galactosidase, and transferriq at 6.5% (w/v) total monomer, ceruloplasmin was also used.

crosslinked gels to achieve measurable values of Rf at monomer concentrations high enough to yield stable gels (13,14). In order to further maximize values of Rf at these conveniently high gel concentrations, the stacking gel buffer was used as the buffer in the resolving gel also. (Since Rf is by definition the ratio of the migration distance of the protein to the displacement of the moving boundary, Rf may also be increased by reducing this displacement. In practice this is achieved by reducing the pH in the resolving gel. This was conveniently effected by using the stacking gel buffer in the resolving gel also). This had the further advantage that some relatively fast moving contaminants of the enzyme remained selectively stacked in the resolving gel. These changes are clearly reflected in Fig. 1. Unstacking of the enzyme was achieved simply by increasing the restrictiveness of the gel. A range of 4.0-7.5% (w/v) total monomer cross-linked with 15% (w/w) iV,N’-diallyltartardiamide (DATD) per combined monomers was found to give values of Rf in the range 0.8 to 0.25. Linear Ferguson plots (Fig. 5) were obtained for each activity state of adenylate cyclase (correlation coefficients 0.9981-0.9998). Since increasing Lubrol concentrations reduced the bandwidth of cyclase (Fig. 2),



Ferguson plot analysis was also conducted at several Lubrol concentrations. Ferguson plots for the enzyme pretreated with glucagon plus Gpp(NH)p determined at 0.01 and 0.1% Lubrol are shown in Fig. 5 and values of KR and Y. in Table II. The enzyme was significantly (P < 0.01) smaller at 0.1% Lubrol and net charge was also decreased. This conclusion was drawn both from direct comparison of the same preparation at different Lubrol concentrations in the same electrophoresis run and from pooling the results of ten experiments at each gel concentration. In order to further establish whether the values of KR obtained in 0.1% Lubrol were characteristic of the smallest active enzyme the effect of harsher disruptive conditions were investigated. High concentrations (1% w/v) of Lubrol did not decrease the size (or polydispersity) of the enzyme (KR = 0.109 +: 0.003). Addition to electrophoresis buffers of the ionic detergents 0.03% ZV-lauryl-sarcosinate (yielding KR = 0.119 f 0.020) or 0.03% deoxycholate (yielding KR = 0.098 + 0.003) in addition to 0.1% Lubrol also failed to reduce the size of the enzyme although they did cause partial loss of enzyme activity. At concentrations of these agents giving up to 80% inactivation no secondary peak of enzyme activity characterized by a smaller molecular radius was observed. A similar conclusion was drawn when urea at concentrations up to 4 M was included in gels in addition to 0.1% Lubrol. 10 :E





% b



3 2 6i


“‘..4,\ t











1% wivl

5. Ferguson plots for solubilized adenylate cyclase pretreated with Gpp(NH)p plus glucagon determined with 0.01% (0) or 0.1% (0) Lubrol PX in the stacking and resolving gels. FIG.










None Gpp(NH)p Gpp(NH)p None Gpp(NH)p Gpp(NH)p

alone + glucagon alone + glucagon

Lubrol (% w/v) concentration 0.10 0.10 0.10 0.01 0.01 0.01



0.100 f 0.010 0.106 0.140

+ 0.006 + 0.006

0.172 +- 0.006 0.154 + 0.004


Molecular radius (nm)

1.4 iz 0.1 1.5 * 0.1

3.6 -t 0.8" 3.8 f 0.8*

1.5 + 0.1 2.8 k 0.2 3.7 + 0.3

4.0 r?; O.&P 4.9 f 0.6'

2.8 + 0.1

5.1 + 0.6’

5.4 f


a Ferguson plots were constructed by using a log-linear regression (6) from values of R, determined at 6 gel concentrations. Data from consecutive experiments on the same material were pooled after testing for statistical homogeneity (6). Values of KX and YO and their standard deviations were obtained from the computerized analysis (6). Molecular radius was calculated by linear interpolation from the standard curve shown as Fig. 7. b The 95% confidence limits for molecular radius were computed (6) taking into account errors in the position of the standard curve and the observations of Ka for each unknown.

Having established the conditions giving rise to the smallest and least polydisperse active enzyme, differences in molecular parameters between the basal and activated states was investigated. The enzyme in three different activity states was examined simultaneously in the same apparatus. Untreated or pretreated soluble fractions from the same batch of membranes were subjected to electrophoresis at six gel concentrations. The entire procedure was repeated. The Ferguson plots of the enzyme in all of the three activation states were indistinguishable (Fig. 6). Molecular


The molecular size of adenylate cyclase in the selected buffer milieu was determined by Ferguson plot analysis as KR and molecular net charge as Yo. The experimental measures of size were then translated to values of molecular radius, fi (Table II), using previously described procedures (6) involving construction of a standard curve, with 10 standard proteins (Fig. 7). The molecular radii of the standard proteins were calculated from their known masses(15) by assumption of sphericity, zero hydration, and a partial specific volume of 0.74. The radii of such standard proteins measured by PAGE are unaffected by nonionic detergent (16). The molecular radius determined for the enzyme in 0.01% Lubrol was 4.9 -+ 0.6 to 5.4 + 0.6 nm (Table II). In 0.1% Lubrol, its geometric mean radius was 3.6 + 0.8 to 4.0 k 0.8 nm.

FIG. 6. Ferguson plots for solubilized adenylate cyclass, either untreated (A), pretreated with Gpp(NH)p alone (0) or pretreated with Gpp(NH)p plus glucagon (0) determined with 0.1% Lubrol PX in the stacking and resolving gels.


This report shows that it is possible to study an intrinsic membrane protein by PAGE in an active conformation. Even in the case of a notoriously labile enzyme such as adenylate cyclase, high recovery of enzymatic activity was achieved. In addition, the problems of large molecular size and low surface charge at neutrality were overcome by a careful choice of buffer system and gel structure. PAGE allows one to measure the size, charge and degree of polydispersity of an enzyme using very small quantities (10-100 pg) of highly impure material. Furthermore, changes in any of these pa-





betweens 0.4 and 1.0 depending on molecular asymmetry (Fig. 4 of Ref. 20). The wide scatter in values of the Stokes radius from gel filtration may be due in part to differences in the detergent concentration. Even the least polydisperse species of the enzyme was more polydisperse than standard non-membrane proteins. Moreover, the activity profiles always showed greater polydispersity at the trailing than at the leading edge, indicating that higher aggregation states are present to some extent under all conditions tested. Whether this degree of polydispersity is typical of intrinsic membrane proteins may only be decided after other such proteins have been characterized in this way. Clearly, the characterization of the enzyme on the basis of its migration distance in PAGE (or in gel filtration or sedimentaMOLECULAR RADIUS lnm, FIG. 7. Standard curve relating KX to molecular tion experiments) is insufficient for answerradius. KX’S were determined by Ferguson plot analying the question whether this is the enzyme sis for bovine serum albumin monomer [lo] and dimer species responsible for function in the mem[16], for thyroglobulin [13], follicle stimulating horbrane. This can be only operationally demone [32], ferritin [44], ceruloplasmin [48], /?-lactofined as the “smallest and least polydisglobulin [54], transferrin [57], catalase [60], and /Iperse” species capable of expressing activgalactosidase [62]. These were then related to their of bandwidth criteria and molecular radii calculated from the known masses (15) ity. Application Ferguson plot analysis have allowed us to by assumption of sphericity, zero hydration and a partial specific volume of 0.74 by a plot of KR”’ vs. pinpoint this species. Failure to detect differences among the molecular radius (6). three activation states of adenylate cyclase rameters brought about by disruptive does not appear to be due to selection of a agents or activation of the enzyme can be separation method of insufficient resolving investigated. The method is capable of rap- capacity. PAGE has been capable of resolvidly handling the large number of samples ing, for example, charge isomers of human such an investigation demands. growth hormone which had been hydroThe active enzyme became smaller and lyzed at 1, 2, or 3 residues out of 191, when less polydisperse with increasing detergent the net conformation of the protein reconcentration, presumably through disag- mained intact due to noncovalent interacgregation. The values of molecular radius tions (21), conformational isomers of the determined for adenylate cyclase in 0.1% same representative protein produced by Lubrol were 3.6 f 0.8 to 4.0 f 0.8 run. These differences in preparative procedures (22) could not be further reduced by high con- and changes in protein structure arising centrations of Lubrol, ionic detergents or from point mutations (23). In the work rechaotropic ions at concentrations which in- ported here, it has clearly been capable of activated most of the enzyme. The values improved resolution of GTPase from adeobtained are considerably smaller than the nylate cyclase compared to previous chrovalues of Stokes radius determined for the matographic methods. Thus the finding of rat liver enzyme previously (4) or for the indistinguishable molecular parameters for enzyme from other sources (17-19). (This basal and activated states of the enzyme is may be explained simply by the molecular likely to reflect true conformational idengeometry of the enzyme, since the ratio of tity. The loss of Gpp(NH)p-response of the geometric mean radius to Stokes radius lies basal enzyme after electrophoresis was not





accompanied by a measurable change in molecular radius. Nonetheless, it is possible that some factor necessary for Gpp(NH)psensitivity, as recently reported (24), has been lost by the enzyme without affecting this parameter significantly. Preparative scale electrophoresis and reconstitution studies are in progress with the hope of clarifying this issue. Throughout this report, no mention was made of the molecular weight of the enzyme. This was omitted since computation of molecular weight from molecular radius requires assumptions with regard to the partial specific volume, degree of detergent binding and hydration and the molecular geometry of the enzyme. None of this information is presently .available for the rat liver enzyme. Even if these parameters are determined experimentally (17), there remain further objections to the calculation of molecular weights from gel filtration and sedimentation velocity data. These are: 1) Uncertainty whether gel filtration measures Stokes radius (20); 2) the impossibility of assigning confidence limits to the values of molecular weight since they are compounded from the relatively large confidence limits (which may or may not be additive) of the three parameters, Stokes radius, partial specific volume and sedimentation coefficient; 3) unavailability of wellcharacterized purified intrinsic membrane proteins which could serve as a set of homogeneous standards; 4) polydispersity of the enzyme. ACKNOWLEDGMENTS We are indebted to David Rodbard for many helpful discussions, provision of computer programs for physical characterization and identity testing by “quantitative PAGE” and for a critical review of the manuscript. A.C.N. is the recipient of a NATO Postdoctoral Fellowship from the Science Research Council of Great Britain. REFERENCES 1. CHRAMBACH, A., JOVIN, T. M., SVENDSEN, P. J., AND RODBARD, D. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), Vol. 2, pp.




27-144, Plenum Press, New York. 2. POHL, S. L., BIRNBAUMER, L., AND RODBELL, M. (1971) J. Biol. Chem. 246, 1849-1856. 3. WELTON, A. F., LAD, P. M., NEWBY, A. C., YAMAMURA, H., NICOSIA, S. AND RODBELL, M. (1977) J. Biol. Cheat. 252, 5947-5950. 4. WELTON, A. F., LAD, P. M., NEWBY, A. C., YAMAMURA, H., NICOSIA, S., AND RODBELL, M.

(1978) Biochim. Biophys. Acta 522,625-639. 5. DIEZEL, W., KOPPERSCHLAEGER, G., AND HOFMANN, E. (1972) Anal. Biochem. 48, 617-620. 6. RODBARD, D., AND CHRAMBACH, A. (1974) in Electrophoresis and Isolectric Focusing on Polyacrylamide Gel (AIlen, R. C., and Maurer, H. R., eds.), pp. 28-61, de Gruyter, Berlin. 7. SALOMON, Y., LONDOS, C., AND RODBELL, M. (1974) Anal. Biochem. 58.541-548. 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 9. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 464-427. 10. JOVIN, T. M., DANTE, M. L., AND CHRAMBACH, A. (1970) Multiophasic Buffer Systems Output, (National Technical Information Service, Springfield, Va. 22151), PB Numbers 196690, 259309 to 259312. 11. JOVIN, T. M. (1973) Biochemistry 12, 871-898. 12. FERGUSON, K. A. (1964) Metabolism 13,985-1002. 13. RODBARD, D., LEVITOV, C., AND CHRAMBACH, A. (1972) Sep. Science 7, 705-723. 14. BAUMANN, G. AND CHRAMBACH, A. (1976) Anal.

Biochem. 70,32-38. 15. SOBER, H. A. (ed.), (1970) Handbook of Biochemistry, Ed. 2, pp. C-10-C-25, Chemical Rubber Co., Cleveland, Ohio. 16. HEARING, V. J., KLINGLER, W. G., EKEL, T. M., AND MONTAGUE, P. M. (1976) Anal. Biochem. 72, 113-122. 17. NEER, E. J. (1974) J. Biol. Chem. 249,6527-6531. 18. HAGA, T., HAGA, K., AND GILMAN, A. G. (1977) J.

Biol. Chem. 252.5776-5782. 19. SWISLOCKI,


J. (1973)


chemistry 12, 1862-1866. 20. RODBARD, D. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), Vol. 2, pp. 145-179, Plenum Press, New York. 21. MUNIZ, N., RODBARD, D., AND CHRAMBACH, A.

(1977) Anal. Biochem. 83, 724-738. 22. SKYLER, J. S., BAUMANN, (1977) Acta Endow. 23. JOHNSON, G. B. (1977) 24. PFUEFFER, T. (1977) 7224-7234.



Suppl. 211, I-40. Genetics 87, 139-157. J. Biol. Chem. 252,

Adenylate cyclase in polyacrylamide gel electrophoresis: solubilized but active.

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