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Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis
Biochimica et BiophysicaActa, 998 (1989)137-144
137
Elsevier BBAPRO33464
Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis Warner S. Simonides and Comelis van Hardeveld Laboratoryfor Physiology, Faculty of medicine, Free University,Amsterdam (The Netherlands)
(Received2 May 1989)
Key words: Parvalbumin;Singlemusclefiber; (X. laevis) The major parvalbnmln~ present in the illofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of th|~ muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dedecyi sulphate (SDS). The criteria used in the identification of proteins as parvalbunfins were: a relative molecular mass (Mr) between |0000 and 14000, an isoelectric point ( p l ) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvallmmins: PAl, M r 14000, p l 4.90; PA2, M r 11000, p l 4.90; PA3, M , 11000, p l 4.95; and PA4, M, 11000, pl 4.25. An nlWavinlet absorhance spectrum d~'actedstic of parvalbunfins was recorded for a purified preparation of these four proteins. Because the apparent M r of rabbit parvalbumin in the gel system used was 14000, whereas the true value is 12100, it h not excluded that the M r of component PAl of 14000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centri|ugafion of tissue homogenates. Analysis of/he protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14000 and 11000 protein bands contained vLvtuallyonly parvalbmnin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of mlnlgels, yielded mean values (lug protein/g wet wC, + S.D.) of 5.2 + 0.8 for nine type 1 fibers, and 1.9 + 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the M r 14000 isoform relative to the combined Mr 11000 isoforms.
Introduction Parvalbun~ns are Ca 2+binding proteins that are expressed in various tissues or distinct cell populations in fish, amphibians and mammals [1,3]. The highest concentrations of parvalbumins are found in skeletal muscle of lower vertebrates, with reported values of up to 1 mmol/kg tissue [2,3]. These proteins share a common ancestry with other Ca 2+ binders, such as troponin-C, myosin light chains and calmodulin, but unlike these the physiological role of parvalbumins is still speculafive. However, several lines of evidence suggest that
Abbreviations: EGTA, ethyleneglycol-bis(/~-aminoethylether)N,N,N',N'.tetraacetic acid; Mr, relativemolecularmass; pl, isoelectric point; SDS,sodiumdodecylsulphate. Correspondence: W.S. Simonides, Laboratory for Physiology,Free University, van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands.
their main function is to increase the speed of muscle relaxation [1]. For instance a close correlation is found in muscle types of various species between the speed of relaxation and the parvalbumin content [4,5]. This correlation remains when the contractile properties of a given muscle are altered as a result of continued stimulation, denervation or cross innervation [5-7]. Although such a function of parvalbumin in mammals has been disputed on the grounds of the low parvalbumin content found in mammalian muscle and the slow rate of Ca 2+ binding to parvalbumin [8], computer simulations have shown that the high parvalbumin concentrations in amphibians and fish could indeed enhance the relaxation rate significantly [9]. But whatever the physiological function, parvalbumin wiU bind C a 2+ when the cytosolic C a 2+ concentration is raised in contracting muscle, releasing in the process some 27 kJ/mol of C a 2+ bound [10]. Given the presence of two Ca 2+ sites per molecule of parvalbumin and the high parvalbumin content of muscles of certain species, this heat release
0167-4838/89/$03.50© 1989 ElsevierSciencePublishersB.V.(BiomedicalDivision)
138 may be of significance in the various stages of heat production during a contraction-relaxation cycle and may contribute to the so called 'unexplained heat' [11,12]. Because of our interest in the considerable differences in the energetics and mechanics between different types of fibers found in skeletal muscle of the African clawed toad Xenopus laevis [13], we wished to determine the parvalbumin content of individual fibers, so as to assess the involvement of parvalbumin more accurately. For the determination of the total parvalbumin content of single fiber homogenates, we examined the possibility of electrophoretic separation of proteins on the basis of molecular weight followed by quantification of the appropriate protein peaks. This required the identification of the possibly multiple isoforms of parvalburain in the iliofibularis muscle which we stucly. Parvalbumins are highly polymorphic and one nmscle may contain several isoforms with different physicochemical properties such as isoelectric point (pI) and relative molecular mass (Mr). Vaz~ations in the latter parameter are of special importance in view of the chosen method of analysis, and for example a total of seven isoforms with Mr values ranging from 10100 to 12200 have been identified in muscles of amphibians (Bufo bufo japonicus [14], Rana catesbeiana [15], Rana esculenta [16] and Rana temporaria [17]). More particular, recent studies on Xenopus laevis using antibodies against parvalbumins, indicate the presence of up to three isoforms, in a single muscle, with Mr values of 12000, 13500 and 14000 [18,19]. In the present study we describe the identification of four isoforms of parvalbumin in the iliofibularis muscle of X. laevis, as well as a method for the quantification of the parvalbumin content of single fibers. The method is illustrated with the analysis of two different types of fiber from the iliofibularis muscle. Materials and Methods
Parvalbumin extraction Freeze.dried fiber bundles or single fibers from the ifiofibularis muscle were homogenized for 3 min in a glass Potter tube with a motor driven pestle in 50 mM phosphate buffer (pH 7.4). Custom-build Potter tubes of 50/fl-5 ml were used depending on the sample size. The homogenate was heated at 60°C for 10 rain and subsequently centrifuged at 10000 × g for 15 rain to pellet cell debris, mitochondria etc. The supernatant, or 'crude extract', containing all soluble proteins was collected and used for analysis without further purification. For practical reasons, small samples, such as single fibers, were usually homogenized directly in solub'dization buffer prior to electrophoresis. This buffer contained 87.5 mM Tris, 2 mM dithiotreitol, 10~ glycerol,
2% sodium dodecyl sulphate (SDS) and 0.001~ bromophenol blue. Homogenization in either phosphate or solubilization buffer yielded the same amount of parvalbumin, although somewhat more high molecular mass proteins were present when using the latter condition. Purified parvalbumins were prepared essentially as described by Strehler etal. [20]. Approx. 4 g of freshly excised iliofibularis muscle were homogenized in 2.5 vol. of 4 mM EDTA (pH 7) for four periods of 30 s using a Sorvall omnimixer. The homogenate was centrifuged at 14 000 x g for 30 rain and the supematant w ~ adjusted to pH 7 with 17~; ammonium hydroxide and heated with stirring at 70 ° C for 30 rain. The suspension was cooled to 10 o C and filtered under reduced pressure through a sintered-glass funnel. The clear filtrate was lyophilized and subsequently dissolved in 400/tl 10 mM sodium acetate (pH 5.7)and dialyzed against the same solution. Centrifugation of the resulting turbid suspension at 60000 x g for 60 min, yielded a clear supernatant containing the parvalbumins.
Electrophoretic analyses Polyacrylamide gel electrophoresis in the presence of SDS was carried out according to Laemmli [21] with a 6.5~ stacking gel and 15~ separating gel, using either a conventional or a minigel apparatus (Bio-Rad). M ~ k c r proteins for the estimation of the Mr were cytochrome c (horse heart) (12400), lysozyme (14000), fl-lactoglobulin (18400), a-chymotrypsin (25000), carbonic anhydrase (30000), bovine serum albumin (68 000) and phosphorylase b (94600) (Bio-Rad or Sigma). Frog parvalbumin (unknown isoform) and rabbit parvalbumin (12100 [22-24]) were obtained from Sigma. Isoelectric focusing electrophoresis was carried out using an LKB multiphore and preformed gels in the pH range of 4.0-6.5 and 3.5-9.5 (LKB). Marker proteins (Bio-Rad) were used to estimate the p l of the proteins. Electrophoresis in the absence of SDS, to determine the mobility of proteins in the presence and absence of Ca 2+, was performed in 8~ gels containing 14 mM Tris and 90 mM glycine (pH 8.6) as described by others [25,26]. Samples were solubilized prior to electrophoresis either in the presence of 2 mM Mg 2+ and 2 mM EGTA, or I mM Ca 2+. Two-dimensional gel electrophoresis following separation in the absence of SDS was carried out by cutting a lane from the gel and placing it on top of an isoelectfic focusing gel (pl determination), or by embedding it in a 6.5~ stacking gel on top of a 15~ separating gel for separation in the presence of SDS. Separation in the second dimension on an SDS gel following isoelectric focusing was as follows. A lane cut from the focusing gel was fixed in 12~ trichloroacetic acid and soaked in water/methanol/acetic acid (5 : 5 : 1, v/v) for I h, then for 10 rain in water, and finally for
139 2 ~h~ in 9 mM Tris-HCl, 2~ SDS, 2~ dithiotreitol (pH 6.8). 'IL~ ~trip was then embedded in a 6.5~ stacking gel on top of a 15~ separating gel for separation in the second dimension in the presence of SDS [21]. All gels were stained with Coomassie brilliant blue R-250.
gel. Measurement of these standards yielded a straight calibration line and the limit of detection with this method is 50 ng protein. The relative standard deviation in the mean parvalbumin content of a typical fiber extract, determined in triplicate, was less than 10~. Results
Quantification of parvalbumin content Single fibers were dissected under dark-field illumination from the iliofibularis muscle. The type of fiber isolated was determined from its position in the muscle and its microscopic appearance according to the criteria developed by ~ e r g r e n and Smith [27]. Following experiments that included force and heat measurements [13], fibers were freeze-dried and weighed using a Calm 29 balance. A crude extract was prepared from these fibers as described above and samples were analyzed by electrophoresis in the presence of SDS using minigels. After electrophoresis, the gel was fixed, stained and dried, and absorption profiles were obtained using an LKB 2202 Ultroscan laser densitometer combined with the LKB 2109-001 gelscan software program for an Apple II. Because the absolute staining intensities varied somewhat from gel to gel, several different known amounts of rabbit parvalbumin were included in each
Identification of parvalbumtns In the analysis of crude muscle extracts we set four criteria, all of which had to be met before a protein was identified as a parvalbumin. These criteria were: an apparent M r between 10 000 and 14000. - an acidic pL Values below 5.0 are commonly found, although pI values of 5.5 have been reported [24]. - a difference between the e|ectrophoretic mobility on native gels (without SDS) in the absence of Ca 2+ (Mg2+-bound form) and presence of Ca 2+ (Ca 2+bound form) [14,15]. - an ultraviolet spectrum characteristic of parvalburains [1-3] with absorption maxiraa in the 250-270 nm region, reflecting the high pe1~'centage of phenylalanine and the absence of tryptophane, and possibly in the 270-280 nm region, due to tyrosine. Gel electrophoretic separation of an iliofibularis extract in the presence of SDS is .~;l~ownin Fig. la. Two -
B
A a
b
c
d
94.6 Q
68£3--
A--
30£3--
iiii~ii!ili,!i~ii~!ili!!~i~!!ii!iii~ill
C~
'0 X
12~4--
bE_ x L.
Fig. 1. Polyacrylamide gel electrophoresis in the presence (A) and absence (B) of SDS. Panel A shows a 155 acrylamide gel containing the following samples: marker proteins with Mr values as indicated (lane a); a crude extract of an iliofibularis muscle, prepared as described in Materials and Methods (lane b); rabbit parvalbumin (lane c); and frog parvalbumin (lane d). Analysis, of this frog preparation using the criteria described in this report indicated that only the fastest moving protein band at approx. Mr 11000 wa~, parvalbumin. Panel 13 shows the effect of Ca 2+ on the mobility of proteins on an 8~ acrylamide gel in 14 mM Tris and 90 mM glycine (pH 8.6) in the absence: of SDS. The following samples were analyzed: a crude extract of an iliofibularis muscle solub'dized in the presence of 2 mM Ca 2+ (lane a); the same extract but solubilized in the absence of Ca 2+, i.e., in the presence of 2 I~MMg 2+ and 2 mM EGTA (lane b); r~t,bbit parvalbumin selubilized in the presence (lane c); and absence of C.a2+ (lane d). Proteins marked A - E showed increased mobility in the ~ibsence of Ca 2+. Migration was from top to bottom, towards the anode. Arrows indicate the corresponding protein bands in lane a and b. This was established by ccmparing protein profiles after electrophoresis and separation of both lanes in the second dimension according to molecubr weight or isoelectric point (see for example Figs. 2 and 3).
140 ED I I
C I
B I
A I
pv
+
~r xl(Sa 8~0--
30.0--
1BA--
12.4--
Fig. 2. Determination of Mr of putative parvalbumins by two-dimensional gel electrophoresis. An extract of an iliofibularis muscle was solubilized in the presence of Ca2+ and separated on a gel in the absence of SDS (see Fig. 1B, lane a). The lane was cut from the gel and embedded in a 6.5% stacking gel on top of a 15% separating gel, both containing SDS [21]. A sample of rabbit parvalbumin (PV) was loaded in a slot alongside the lane. Positions of marker proteins are indicated. Arrows indicate the proteins that correspond to bands A, B and D.
prominent protein bands can be seen towards the front of the gel, comigrating with frog and rabbit parvalbumin. Comparison with marker proteins in this example and five other gels indicated average apparent M r values for the two protein bands of 14000 4. 200 and
] 1000 4- 500 (means 5: S.D.) (the heavier component comigrated with lysozyme, M r 14000, not shown). The same extract was then subjected to electrophoresis in the absence of SDS at p H 8.6, which gives separation of (acidic) proteins, including parvalbumins [14,15], according to size, conformation and net negative charge (Fig. lb). When run in the presence of Ca 2+, several protein bands could be distinguished. Five of these, marked A - E , showed increased mobility in the absence of Ca 2+ and presence of Mg 2+, as did rabbit parvalbumin. In one experiment (not shown) we included troponin C and myosin light chains (Sigma). Both proteins comigrated with component E and showed a similar, slightly higher mobility in the absence of Ca 2+. To test which Of the five protein bands corresponded to the two low Mr bands seen in Fig. la, we separated a gel as in Fig. l b (lane a ) i n the second dimension in the presence of SDS. This is shown in Fig. 2, where the major component B comigrates with rabbit parvalbumin and components A and D migrate to an apparent M r of 11000. Component C has an M r of 30000, and component E is split up in two proteins of M r 18000 and 19000. With components A, B and D meeting two of the criteria for parvalbumins, a gel lane as in Fig. l b (lane a) was then run in the second dimension on an isoelectri¢ focusing gel to determine the p I values of these proteins. As shown in Fig. 3, component A proved to be composed of two proteins with slightly different pI; 4.90 and 4.95, whereas a single band was found for component B ( p I 4.90) and component D ( p I 4.25). Component E (two proteins were discernible already in
pH
4.55.-
4.ssF
~-pv 5.10--
6,00--
Fig. 3. Determination of pl of putative parvalbumins by two-dimensional gel electrophoresis. An extract of an iliofibularis muscle was solubilized in the presence of Ca2+ and separated on a gel in the absence of SDS (see Fig. 1B, lane a). The lane was cut from me gel and placed on top and in the middle of a preformed pH gradient gel (pH 4.0-6.5 (LKB)). The lane is depicted alongside the gel for clarity. Marker proteins with known pl values are visible at beth sides of the gel and a rabbit parvalbumin sample is seen on the far right (PV). Large arrows mark the protein bands at pH 4.90 and 4.95 (component A), 4.90 (component B) and 4.25 (component D). The small arrow indicates two closely spaced bands corresponding to component E.
141 A a
b
B c
161
TABLE I
Properties of parvalbuminsfrom the iliofibularismuscle of X. laevis Apparent relative molecular mass (Mr) was estimated by electrophoresis in the presence of SDS (see Figs. 1 aI~d 2~, and isoelectric point (pl) by isoelectric focusing (see Figs. 3 and 5). These parameters were determined on at least three separate occasions and relative standard deviations were less than 55 for the M r values and about 15 for the p l values.
A--
B--
7 ,,~2/0
Mr pl
!
2 7 0I nm
2 9 I0
Fig. 4. Polyacrylamide gel electrophoresis in the absence of SDS of purified parvalbumins (A) and ultraviolet absorbance spectrum of this preparation (B). Parvalbumin.~ were purified from an iliofibularis muscle as described in Materials and Methods. (A) Samples of the preparation were solubilized in the presence (lane b) or absence of Ca 2+ (lane c) and separated on a gel as described for Fig. lB. Lane a shows a sample of the crude extract from which the parvalbumins were isolated, solubilized in the absence of Ca 2+. (]8) The absorbance spectrum of the parvalbmnin preparation, dissolved in 1~ mM sodium acetate (pH 5.7) at a protein concentration of 5 mg/ml, wa~ recorded using a cuvette with a l-cm light path. The absorbance fel~ ~vithout interruption to a baseline level for wavelengths above 280 ram.
the first dimension in this example) yielded two closely spaced bands around p I 4.5. Rabbit parvalbumin had a pI of 4.90 in agreement with published values [22]. Parvalbumins were then extracted from an iliofibuiaris muscle and purified as described in Materials and Methods. The final preparation was composed for more than 955 of the Mr 14000 and 11000 proteins, the only slight contamination being an Mr 68000 protein. Fig. 4a shows this preparation and the extract from which it was isolated, separated on a gel in the absence of SDS. The three major bands correspond to the components A, B and D tentatively identified above as parvalbumins. The fourth, minor band corresponds to the contaminating Mr 68000 protein. Using this preparation, an ultraviolet spectrum was recorded which showed an absorption profile characteristic of parvalbumins (Fig. 4b). Taken together, these results show that the Mr 14000 and 11000 bands in the crude muscle extract contain a total of four different proteins meeting the criteria for identification as a parvalbumin (Table I). However, the data presented above do not exclude the possible presence of a significant amount of proteins other than parvalbumin in the respective bands, which would preclude the quantification of parvalbumin content of crude
PAl
PA2
PA3
PA4
14000 4.90
11000 4.90
I1000 4.95
11000 4.25
extracts using SDS gels. In order to check whether the Mr 14000 and 11000 bands indeed contain only the acidic proteins identified above, muscle extracts were first separated on an isoelectric focusing gel and then run in the second dimension on an SDS gel. The results in Fig. 5 of a pH 4.0-6.5 gel, show that no proteins other than the identified parvalbumins have an Mr value below 14000, with the possible exception of one acidic protein (pI 4.0), that migrated to an Mr of 11000, and a very small amount of protein visible as a faint spot at M, 14000, p l 4.5. Proteins with M r > 30000 that do not show up on the gel in Fig. 5 had p l values between 7 and 9. This was checked on a pH 3.5-9.5 gel, and also in this pH range we could not detect any proteins of M, < 14 000 (unpublished data). Finally, we checked the possibility that parvalbumin was lost in the pellet that was discarded after extraction pH 4.B5 I
5.10
6.00
I
I
680-
'0
3o0-
(.
12A-
Fig. 5. Two-dimensional gel electrophoresis of a crude muscle extract. A crude extract of an iliofibul~'is muscle was f'wst run on a pH 4.0-6.5 isoelectric focusing gel (LKB) and then in the second dimension in the presence of SDS as described in Materials and Methods. A sample of the crude extract was run on the gel alongside the isoelectric focusing strip. Arrows indicate two unidentified spots at Mr 14000 and 11000. The smear of stain to the right of the parvalbumin spots on this gel is probably caused by diffusion of protein in the interface between the gel strip and the separating gel. This was not observed on other occasions.
B
A m b a
b
c
d
e
f
0
h
c
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M r xl[~3 Fig. 6. Polyacrylamidegel electrophoresis using mlnigels. (A) A 15% minigel containing a crude extract of an iliofibularis muscle (lane a), rabbit (lane b) and a sample of the purified parvalbumin preparation (lane c) (see also Fig. 4). (B) A gradient minigel (10-20%) showing the separation of crude extracts of three type 1 fibers (lanes c, d and e) and rabbit parvalbumin (all other lanes). The equivalent of dry weight of ~e fibers that was loaded on the gel was 27/tg for lane c and 19 ttg for lanes d and e. The amounts of rabbit parvalbumin on the gel ranged from 0.25 to 1.25 ltg protein. The position of the M r 14000 and 11000 components is indicated° (C) Absorbance profile of the lower part of lane c ob'a6ned by laser densitometry.Peak areas correspond to 0.45 Itg protein (Mr 14000) and 0.18/tg protein (Mr llO00). The sample in lane d contained 0.24 and 0.08/tg, and the one in lane e contained 0.22 and 0.07 Itg of the Mr 14000 and 11000 components, respectively.
parvalbumin
TABLE II
Parvalbumin content of single fibers of the iliofibularis muscle of X. laevis Sinsle fibers were dissected from freeze-dried iliofibularis muscle and classified according to the criteria in Ref. 27. Fibers were homogenized in solub'dizatiun buffer and crude extracts were separated on minigels (see also Fig. 6). Protein profiles of stained and dried gels were obtained by laser densitometry and peak areas were calculated by computer. Data represent the mean amount (4- S.D.) of Mr 14000 (PAl) and combined Mr 11000 parvalbuming (PA2-4) present in fibers, expressed as mg protein/g fiber wet wt. (fiber wet wt. = 3.73< fiber dry wt.). The number of fibers analyzed is shown in parentheses. Two of the five examined type 2 fibers had undetectable levels of the Mr 11000 isoforms. Differences in the parvalbumin content (total and PAl or PA2-4) between type 1 and type 2 fibers were significant; 2P < 0.005 (Student's t-test).
PAl PA24
TYPE 1
TYPE 2
3.804-0.50 (9) 1.434-0.35 (9)
1.544-0.68 (5) 0.54 4-0.27 (3)
of the soluble fraction. However, extensive solubilization of pellet material followed b y electrophoresis, indicated that no proteins of Mr 14000 or 11000 were present in this fraction.
Quantification of parvalbumin in single muscle fibers Relatively fast (type 1) a n d slow (type 2) contracting and relaxing fibers were isolated from the iliofibularis muscle, a n d extracts were obtained using solub'llization buffer as homogenization medium, as described in Materials a n d Methods. Fig. 6a and b shows examples of a whole muscle extract a n d several single fiber extracts separated o n minigels together with r a b b i t p a r v a l b u m i n a n d the purified parvalbumin p r e p a r a t i o n of Fig. 4. G r a d i e n t gels of 10-20~; acrylamide were initially used, b u t linear 1 5 $ gels yielded similar results and these were routinely used. Fiber d r y weights ranged from 2 0 - 8 0 ltg, and the equivalent of 1 5 - 3 0 Itg was usually enough to obtain a reliable absorbance reading
143 of the parvalbumins from the gel. An example of a laser scan of the low Mr range of a fiber extract is given in Fig. 6c. Table II sums up the average values of the parvalbumin content of type 1 and type 2 fibers. The data show that the Mr 14000 isoform is by far the most abundant in both fiber types and in fact two of the examined type 2 fibers did not yield detectable amounts of the low molecular weight parvalbumins. The total parvalbumin content of the fast type 1 fibers wa~ nearly 3-fold higher than that of the type 2 fibers. Discussion In the present study we examined the possibility of determinin~ the parvalbumin content of single muscle fibers from the ilio~bularis muscle of X. laevis, using protein profiles of crude extracts. Which protein bands after gel electrophoresis correspond to parvalbumin had to be assessed first, because although the Ca2+binding property of parvalbumin has been conserved for over 500 million years of evolution, a large number of isoforms with different physicochemical properties has evolved [1-3]. This polymorphic character of parvalbuwjns is again exemplified by the present results, with four proteins identified in the iliofibularis muscle that met the criteria for parvalbnmin. Our data may be compared to recently published results from a study in which polyclonal antibodies against parvalbumins were used to identify parvalbumins in various muscles of X. laevis [19]..'~u'ee immunoreactive protein bands were f,~und after gel electrophoresis according to Laemmli [2!] at M r 12000, 13 500 and 14000 in sartorius and mylohyoid muscle extracts. Gastrocnemius extracts contained only the 12000 and 14000 species. Given the limited accuracy of M r determinations on gels, our data with one M, 14000 component and one group of M, 11000 components, would seem in good agreement with this latter situation. In Fig. la, the three low M, isoforms comigrated with a frog parvalbumin (unidentified isoform, Sigma). Frog parvalbumins have published M r values ranging from 10000 to 12000 [14-17], and it is likely that the group of proteins correspond to the immunoreactive species at M, 12000 in ReL 19. The major isoform in our study (PAl) co-migrated, as one parvalbn~min species did in Ref. 19, with lysozyme ( M r 14000). This is a relatively high M, value for parvalbumin, but nonetheless, PAl co-migrated with rabbit parvalbumin, which has an M r of 12100 as calculated from the amino acid composition [23]. This obvious overestimation of the M, value may be related to the electrophoresis conditions of Laemmli [21] which were used both by Schwartz and Kay [19] and by us, whereas other methods have been used in all previous studies with rabbit parvalbumin (see for example Ref. 22). Given the particular behavior of rabbit
parvalbumin in this gel system, a true Mr value of around 12000 for the major parvalbumin in X. laevis muscle can not be excluded and is perhaps more likely, since the M r of all parvalbumins thus far characterized does not exceed 12 200. The results have shown that the Mr 14000 and 11000 bands on SDS gels are virtually completely composed of parvalbumins, and that no parvalbumin is lost in the preparation of the extract. The small amount of protein with p I around 4.0 and M r 11000, that was observed once on an isoelectric focusing gel (Fig. 5), may represent a fifth parvalbumin that was not detected in the other analyses. The same may hold true for the acidic protein (pl 4.5) just visible at M, 14000 in Fig. 5. Although the identity of these contaminating proteins is uncertain, their contribution to the parvalbumin peaks" is negli~ole and we eventually chose SDS gels for the quantification. Separation on gels without SDS, although also allowing the separate determination of PA4, was not used, because frequent tailing of the protein bands on such gels prevented accurate measurements. Concerning the absolute parvalbumin content of the fibers, previous studies give a maximal parvalbumin content of frog muscle of 3.5 mg/g wet wt. [14,15,17]. The present results show that due to the heterogeneous composition of the muscle fiber population, higher parvalbumin concentrations can occur in individual cells, which is line with the reported range of parvalbumin contents in different fiber types in mammalian muscle, assessed using immunohistochemical techniques [1,4]o The data in Table II give an average content in type 1 fibers of 5.2 mg/g wet wt., and values as high as 7.0 mg/g wet wt. have been found, which equals 0.59 /tmol/g wet wt., assuming a true Mr of 12 000 for the compone.~ PAl. On the other hand, the lowest parvall-am~ content that we encountered in type 2 fibers was 0.09/~mol/g wet wfL. This broad range of parvalbumin contents, when determined in conjunction with force and heat measurements, as is currently done in our laboratory, should enable a better evaluation of the involvement of parvalbumin in the dynamics and energetics of contracting muscle. With respect to this, the higher parvalbumin content of type 1 as compared to type 2 fibers (Table II) is at least in qualitative agreement with the proposed role of parvalbumin in muscle relaxation [1], since the average initial relaxation rate of the type 1 fibers used in the present study was 33~ higher than that of the type 2 fibers (unpublished obse~ation). Although a thorough characterization of the identified parvalbumins is beyond the scope of this study, some conclusions may be drawn from the obtained data. On the basis of differences in amino acid composition of the isoforms thus far characterized, two genetic lineages, a and fl, have been identified. A higher content of acidic residues in isoforms of fl lineage results in
144 p l values of less than 4.8, whereas more alkaline values are exclusively found in type a parvalbumins [14,15]. Classification of the four parvalbumins found in the iliofibularis on the basis of this criterium, would suggest that three of these are of a lineage (pI > 4.8 [14]): PAl, the most abundant isoform with (apparent) M r 14000 and pI 4.90; PA2, M r 11000 and p l 4.90; and PA3, Mr 11000 and p l 4.95. PA4, the third Mr 11000 isoform, with a p l of 4.25, clearly would be of t-lineage. The identification of this single fl isoform substantiates the already predicted presence of such an isoform on the basis of previous work [18]. In that study, expression of a cDNA sequence encoding a parvalbumin from X. laevis, yielded a protein of Mr 12 000 that showed a striking 76~ homology with carp fl parvalbumin of pI 4.25 [18,28]. Like the carp parvalbumin, this fi isoform lacked tyrosine [18,28], which is present in most fi lineage proteins, but absent in virtually all a isoforms. Because PAl-3 are identified as a isoforms, it is unexpected that we find a strong absorbance around 280 nm characteristic of tyrosine in the ultraviolet spectrum of Fig. 4. Furthermore, when compared with pubfished spectra, the relative peak heights due to phenylalanine (253-268 nm) and tyrosine suggest that the greater portion of the parvalbumin in our preparation contains tyrosine [14,15,17,20]. These data may be reconciled by assuming that at least PAl, the major component in the preparation is closely related to the particular a isoform found in Rana temporaria (pl 4.97) [17] and chicken muscle (pI 4.9) [20]. The amino acid compositions of these proteins is virtually identical and separates them from other a lineage parvalbumins by the presence of one tyrosine residue. The spectrum of Fig. 4 indeed shows a close resemblance to those pubfished for these parvalbumins [17,20]. Comparison of published data on the effect of Ca 2+ binding on the electrophoretic mobility [14,15] and those in Figs. 1 and 4, gives no indication of a correlation between genetic lineage, molecular weight or p I and the extent of this electrophoretic effect. No extra information is therefore obtained from these results, but the striking differences in the magnitude of the effect, which is thought to be related to conformational changes, suggest considerable structural differences between the various isoforms. Given this variation it may at least be concluded that components PA2 and PA3 are very closely related, as they showed identical mobility in the Ca 2+ bound and Mg 2+ bound form. In summary, the described method allows a quantitative analysis of the large differences in total parvalbumin content between individual muscle fibers. Regarding the identification of parvalbumins, our results extend the previously published data on X. laevis muscles [19], by showing that in the iliofibularis muscle the protein fraction of Mr 11000 contains three isoforms of parvalbumin and that the M r 14000 fraction comprises
the major parvalbumin isoform with a probable true Mr of approx. 12 000.
Acknowledgement The excellent technical assistance of Mr. R. Zaremba is gratefully acknowledged.
References 1 Heizmann, C.W. (1984) Experientia 40, 910-921. 2 Wnuk, W., Cox, J.A. and Stein, E.A. (1982) in Molecular Biology. An International Series of Monographs and Textbooks. Calcium and Cell Function, 2 (Cheung, W.Y., ed.), pp. 243-278, Academic Press, New York. 3 Demaille, J., Dutruge, E., Capony, J.P. and Pech~re, J.F. (1974) in Calcium-binding Proteins (Drabikowski, W., Strzelecka-Golaszewska, H. and Carafoli, E., eds.), pp. 643-677, Elsevier/NorthHolland, Amsterdam. 4 Cello, M.R. and Heizmann, C.W. (1982) Nature 297, 504-506. 5 Heizmann, C.W., Berchtuld, M.W. and Rowlerson, A.M. (1982) Biochemistry 79, 7243--7247. 6 Miintener, M., Rowlerson, A.M., Berchtold, M.W. and Heizmann, C.W. (1987) J. Biol. Chem. 262, 465-469. 7 Klug, G.A., Leberer, E., Leisner, E., Simoneau, J.A. and Pette, D. (1988) Pfliigers Arch. 411, 126-131. 8 Johnson, J.D., Robinson, D.E. and Robertson, S.P. (1981) in The Regulation of Muscle Contraction: Excitation-Contraction Coupling (Grinell, A.D. and Brazier, M.A.B., eds.), pp. 241-259, Academic Press, New York. 9 Gillis, LM., Thomason, D., Lef~vre, J. and Kret~inger, R.H. (1982) J. Muscle Res. Cell Motil. 3, 377-398. 10 Tanokura, M. and Yamada, K. (1987) Biochemistry 26, 7668-7674. 11 Curtin, N.A. and Woledge, R.C. (1982) Fed. Proc. 41, 161-162. 12 Curtin, N.A. and Woledge, R.C. (1978) Physiol. Rev. 58, 690-761. 13 F]Tinga, G., Lgmnergren, J. and Stienen, G.J.M. (1987) J. Physiol. 393, 399-412. 14 Tanokura, M., Goto, K., Toyomori, Y. and Yamada, K. (1987) J. Biochem. 102, 1133-1139. 15 Tanokura, M., Aramaki, H., Goto, K., Hashimoto, U., Toyomori, Y. and Yamada, K. (1986) J. Biochem. 99, 1211-1218. 16 Capony, J.P., Demaille, J., Pina, C. and Pech~re, LF. (1975) Eur. J. Biochem. 56, 215-227. 17 Gosselin-Rey, C. and Gerday, C. (1977) Biochim. Biophys. Acta 492, 53-63. 18 Kay, B.K., Shah, A.J. and Halstead, W.E. (1987) J. Cell Biol. 104, 841-847. 19 Schwartz, L.M. and Kay, B.K. (1988) Dev. Biol. 128, 441-452. 20 Strehler, E.E., Eppenberger, H.M. and H"e:zmann, C.W. (1977) FEBS Lett. 78, 127-133. 21 Laemmli, U.K. (1970) Nature 227, 680-685. 22 Blum, H.E., Lehky, P., Kohler, L., Stein, E.A. and Fischer, E.H. (1977) J. Biol. Chem. 252, 2834-2838. 23 Enfield, D.L., Ericsson, L.H., Blum, H.E., Fischer, E.H. and Neurath, H. (1975) Proc. Natl. Acad. Sci. USA 72, 1309-1313. 24 Capony, J.P., Pina, C. and Pech~re, J.F. (1976) Eur. J. Biochem. 70, 123-135. 25 Tanokura, M., Tawada, Y., Ono, A. and Ohtsuki, I. (1983) J. Biochem. 93, 331-337. 26 Head, J.F. and Perry, S.V. (1974) Biochem. J. 197, 145-154. 27 Lannergren, J. and Smith, R.S. (1966) Acta Physiol. Scand. 68, 2263-2274. 28 Coffee, C.J. and Bradshaw, R.A. (1973) J. Biol. Chem. 248, 3305-3312.
137
Elsevier BBAPRO33464
Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis Warner S. Simonides and Comelis van Hardeveld Laboratoryfor Physiology, Faculty of medicine, Free University,Amsterdam (The Netherlands)
(Received2 May 1989)
Key words: Parvalbumin;Singlemusclefiber; (X. laevis) The major parvalbnmln~ present in the illofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of th|~ muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dedecyi sulphate (SDS). The criteria used in the identification of proteins as parvalbunfins were: a relative molecular mass (Mr) between |0000 and 14000, an isoelectric point ( p l ) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvallmmins: PAl, M r 14000, p l 4.90; PA2, M r 11000, p l 4.90; PA3, M , 11000, p l 4.95; and PA4, M, 11000, pl 4.25. An nlWavinlet absorhance spectrum d~'actedstic of parvalbunfins was recorded for a purified preparation of these four proteins. Because the apparent M r of rabbit parvalbumin in the gel system used was 14000, whereas the true value is 12100, it h not excluded that the M r of component PAl of 14000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centri|ugafion of tissue homogenates. Analysis of/he protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14000 and 11000 protein bands contained vLvtuallyonly parvalbmnin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of mlnlgels, yielded mean values (lug protein/g wet wC, + S.D.) of 5.2 + 0.8 for nine type 1 fibers, and 1.9 + 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the M r 14000 isoform relative to the combined Mr 11000 isoforms.
Introduction Parvalbun~ns are Ca 2+binding proteins that are expressed in various tissues or distinct cell populations in fish, amphibians and mammals [1,3]. The highest concentrations of parvalbumins are found in skeletal muscle of lower vertebrates, with reported values of up to 1 mmol/kg tissue [2,3]. These proteins share a common ancestry with other Ca 2+ binders, such as troponin-C, myosin light chains and calmodulin, but unlike these the physiological role of parvalbumins is still speculafive. However, several lines of evidence suggest that
Abbreviations: EGTA, ethyleneglycol-bis(/~-aminoethylether)N,N,N',N'.tetraacetic acid; Mr, relativemolecularmass; pl, isoelectric point; SDS,sodiumdodecylsulphate. Correspondence: W.S. Simonides, Laboratory for Physiology,Free University, van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands.
their main function is to increase the speed of muscle relaxation [1]. For instance a close correlation is found in muscle types of various species between the speed of relaxation and the parvalbumin content [4,5]. This correlation remains when the contractile properties of a given muscle are altered as a result of continued stimulation, denervation or cross innervation [5-7]. Although such a function of parvalbumin in mammals has been disputed on the grounds of the low parvalbumin content found in mammalian muscle and the slow rate of Ca 2+ binding to parvalbumin [8], computer simulations have shown that the high parvalbumin concentrations in amphibians and fish could indeed enhance the relaxation rate significantly [9]. But whatever the physiological function, parvalbumin wiU bind C a 2+ when the cytosolic C a 2+ concentration is raised in contracting muscle, releasing in the process some 27 kJ/mol of C a 2+ bound [10]. Given the presence of two Ca 2+ sites per molecule of parvalbumin and the high parvalbumin content of muscles of certain species, this heat release
0167-4838/89/$03.50© 1989 ElsevierSciencePublishersB.V.(BiomedicalDivision)
138 may be of significance in the various stages of heat production during a contraction-relaxation cycle and may contribute to the so called 'unexplained heat' [11,12]. Because of our interest in the considerable differences in the energetics and mechanics between different types of fibers found in skeletal muscle of the African clawed toad Xenopus laevis [13], we wished to determine the parvalbumin content of individual fibers, so as to assess the involvement of parvalbumin more accurately. For the determination of the total parvalbumin content of single fiber homogenates, we examined the possibility of electrophoretic separation of proteins on the basis of molecular weight followed by quantification of the appropriate protein peaks. This required the identification of the possibly multiple isoforms of parvalburain in the iliofibularis muscle which we stucly. Parvalbumins are highly polymorphic and one nmscle may contain several isoforms with different physicochemical properties such as isoelectric point (pI) and relative molecular mass (Mr). Vaz~ations in the latter parameter are of special importance in view of the chosen method of analysis, and for example a total of seven isoforms with Mr values ranging from 10100 to 12200 have been identified in muscles of amphibians (Bufo bufo japonicus [14], Rana catesbeiana [15], Rana esculenta [16] and Rana temporaria [17]). More particular, recent studies on Xenopus laevis using antibodies against parvalbumins, indicate the presence of up to three isoforms, in a single muscle, with Mr values of 12000, 13500 and 14000 [18,19]. In the present study we describe the identification of four isoforms of parvalbumin in the iliofibularis muscle of X. laevis, as well as a method for the quantification of the parvalbumin content of single fibers. The method is illustrated with the analysis of two different types of fiber from the iliofibularis muscle. Materials and Methods
Parvalbumin extraction Freeze.dried fiber bundles or single fibers from the ifiofibularis muscle were homogenized for 3 min in a glass Potter tube with a motor driven pestle in 50 mM phosphate buffer (pH 7.4). Custom-build Potter tubes of 50/fl-5 ml were used depending on the sample size. The homogenate was heated at 60°C for 10 rain and subsequently centrifuged at 10000 × g for 15 rain to pellet cell debris, mitochondria etc. The supernatant, or 'crude extract', containing all soluble proteins was collected and used for analysis without further purification. For practical reasons, small samples, such as single fibers, were usually homogenized directly in solub'dization buffer prior to electrophoresis. This buffer contained 87.5 mM Tris, 2 mM dithiotreitol, 10~ glycerol,
2% sodium dodecyl sulphate (SDS) and 0.001~ bromophenol blue. Homogenization in either phosphate or solubilization buffer yielded the same amount of parvalbumin, although somewhat more high molecular mass proteins were present when using the latter condition. Purified parvalbumins were prepared essentially as described by Strehler etal. [20]. Approx. 4 g of freshly excised iliofibularis muscle were homogenized in 2.5 vol. of 4 mM EDTA (pH 7) for four periods of 30 s using a Sorvall omnimixer. The homogenate was centrifuged at 14 000 x g for 30 rain and the supematant w ~ adjusted to pH 7 with 17~; ammonium hydroxide and heated with stirring at 70 ° C for 30 rain. The suspension was cooled to 10 o C and filtered under reduced pressure through a sintered-glass funnel. The clear filtrate was lyophilized and subsequently dissolved in 400/tl 10 mM sodium acetate (pH 5.7)and dialyzed against the same solution. Centrifugation of the resulting turbid suspension at 60000 x g for 60 min, yielded a clear supernatant containing the parvalbumins.
Electrophoretic analyses Polyacrylamide gel electrophoresis in the presence of SDS was carried out according to Laemmli [21] with a 6.5~ stacking gel and 15~ separating gel, using either a conventional or a minigel apparatus (Bio-Rad). M ~ k c r proteins for the estimation of the Mr were cytochrome c (horse heart) (12400), lysozyme (14000), fl-lactoglobulin (18400), a-chymotrypsin (25000), carbonic anhydrase (30000), bovine serum albumin (68 000) and phosphorylase b (94600) (Bio-Rad or Sigma). Frog parvalbumin (unknown isoform) and rabbit parvalbumin (12100 [22-24]) were obtained from Sigma. Isoelectric focusing electrophoresis was carried out using an LKB multiphore and preformed gels in the pH range of 4.0-6.5 and 3.5-9.5 (LKB). Marker proteins (Bio-Rad) were used to estimate the p l of the proteins. Electrophoresis in the absence of SDS, to determine the mobility of proteins in the presence and absence of Ca 2+, was performed in 8~ gels containing 14 mM Tris and 90 mM glycine (pH 8.6) as described by others [25,26]. Samples were solubilized prior to electrophoresis either in the presence of 2 mM Mg 2+ and 2 mM EGTA, or I mM Ca 2+. Two-dimensional gel electrophoresis following separation in the absence of SDS was carried out by cutting a lane from the gel and placing it on top of an isoelectfic focusing gel (pl determination), or by embedding it in a 6.5~ stacking gel on top of a 15~ separating gel for separation in the presence of SDS. Separation in the second dimension on an SDS gel following isoelectric focusing was as follows. A lane cut from the focusing gel was fixed in 12~ trichloroacetic acid and soaked in water/methanol/acetic acid (5 : 5 : 1, v/v) for I h, then for 10 rain in water, and finally for
139 2 ~h~ in 9 mM Tris-HCl, 2~ SDS, 2~ dithiotreitol (pH 6.8). 'IL~ ~trip was then embedded in a 6.5~ stacking gel on top of a 15~ separating gel for separation in the second dimension in the presence of SDS [21]. All gels were stained with Coomassie brilliant blue R-250.
gel. Measurement of these standards yielded a straight calibration line and the limit of detection with this method is 50 ng protein. The relative standard deviation in the mean parvalbumin content of a typical fiber extract, determined in triplicate, was less than 10~. Results
Quantification of parvalbumin content Single fibers were dissected under dark-field illumination from the iliofibularis muscle. The type of fiber isolated was determined from its position in the muscle and its microscopic appearance according to the criteria developed by ~ e r g r e n and Smith [27]. Following experiments that included force and heat measurements [13], fibers were freeze-dried and weighed using a Calm 29 balance. A crude extract was prepared from these fibers as described above and samples were analyzed by electrophoresis in the presence of SDS using minigels. After electrophoresis, the gel was fixed, stained and dried, and absorption profiles were obtained using an LKB 2202 Ultroscan laser densitometer combined with the LKB 2109-001 gelscan software program for an Apple II. Because the absolute staining intensities varied somewhat from gel to gel, several different known amounts of rabbit parvalbumin were included in each
Identification of parvalbumtns In the analysis of crude muscle extracts we set four criteria, all of which had to be met before a protein was identified as a parvalbumin. These criteria were: an apparent M r between 10 000 and 14000. - an acidic pL Values below 5.0 are commonly found, although pI values of 5.5 have been reported [24]. - a difference between the e|ectrophoretic mobility on native gels (without SDS) in the absence of Ca 2+ (Mg2+-bound form) and presence of Ca 2+ (Ca 2+bound form) [14,15]. - an ultraviolet spectrum characteristic of parvalburains [1-3] with absorption maxiraa in the 250-270 nm region, reflecting the high pe1~'centage of phenylalanine and the absence of tryptophane, and possibly in the 270-280 nm region, due to tyrosine. Gel electrophoretic separation of an iliofibularis extract in the presence of SDS is .~;l~ownin Fig. la. Two -
B
A a
b
c
d
94.6 Q
68£3--
A--
30£3--
iiii~ii!ili,!i~ii~!ili!!~i~!!ii!iii~ill
C~
'0 X
12~4--
bE_ x L.
Fig. 1. Polyacrylamide gel electrophoresis in the presence (A) and absence (B) of SDS. Panel A shows a 155 acrylamide gel containing the following samples: marker proteins with Mr values as indicated (lane a); a crude extract of an iliofibularis muscle, prepared as described in Materials and Methods (lane b); rabbit parvalbumin (lane c); and frog parvalbumin (lane d). Analysis, of this frog preparation using the criteria described in this report indicated that only the fastest moving protein band at approx. Mr 11000 wa~, parvalbumin. Panel 13 shows the effect of Ca 2+ on the mobility of proteins on an 8~ acrylamide gel in 14 mM Tris and 90 mM glycine (pH 8.6) in the absence: of SDS. The following samples were analyzed: a crude extract of an iliofibularis muscle solub'dized in the presence of 2 mM Ca 2+ (lane a); the same extract but solubilized in the absence of Ca 2+, i.e., in the presence of 2 I~MMg 2+ and 2 mM EGTA (lane b); r~t,bbit parvalbumin selubilized in the presence (lane c); and absence of C.a2+ (lane d). Proteins marked A - E showed increased mobility in the ~ibsence of Ca 2+. Migration was from top to bottom, towards the anode. Arrows indicate the corresponding protein bands in lane a and b. This was established by ccmparing protein profiles after electrophoresis and separation of both lanes in the second dimension according to molecubr weight or isoelectric point (see for example Figs. 2 and 3).
140 ED I I
C I
B I
A I
pv
+
~r xl(Sa 8~0--
30.0--
1BA--
12.4--
Fig. 2. Determination of Mr of putative parvalbumins by two-dimensional gel electrophoresis. An extract of an iliofibularis muscle was solubilized in the presence of Ca2+ and separated on a gel in the absence of SDS (see Fig. 1B, lane a). The lane was cut from the gel and embedded in a 6.5% stacking gel on top of a 15% separating gel, both containing SDS [21]. A sample of rabbit parvalbumin (PV) was loaded in a slot alongside the lane. Positions of marker proteins are indicated. Arrows indicate the proteins that correspond to bands A, B and D.
prominent protein bands can be seen towards the front of the gel, comigrating with frog and rabbit parvalbumin. Comparison with marker proteins in this example and five other gels indicated average apparent M r values for the two protein bands of 14000 4. 200 and
] 1000 4- 500 (means 5: S.D.) (the heavier component comigrated with lysozyme, M r 14000, not shown). The same extract was then subjected to electrophoresis in the absence of SDS at p H 8.6, which gives separation of (acidic) proteins, including parvalbumins [14,15], according to size, conformation and net negative charge (Fig. lb). When run in the presence of Ca 2+, several protein bands could be distinguished. Five of these, marked A - E , showed increased mobility in the absence of Ca 2+ and presence of Mg 2+, as did rabbit parvalbumin. In one experiment (not shown) we included troponin C and myosin light chains (Sigma). Both proteins comigrated with component E and showed a similar, slightly higher mobility in the absence of Ca 2+. To test which Of the five protein bands corresponded to the two low Mr bands seen in Fig. la, we separated a gel as in Fig. l b (lane a ) i n the second dimension in the presence of SDS. This is shown in Fig. 2, where the major component B comigrates with rabbit parvalbumin and components A and D migrate to an apparent M r of 11000. Component C has an M r of 30000, and component E is split up in two proteins of M r 18000 and 19000. With components A, B and D meeting two of the criteria for parvalbumins, a gel lane as in Fig. l b (lane a) was then run in the second dimension on an isoelectri¢ focusing gel to determine the p I values of these proteins. As shown in Fig. 3, component A proved to be composed of two proteins with slightly different pI; 4.90 and 4.95, whereas a single band was found for component B ( p I 4.90) and component D ( p I 4.25). Component E (two proteins were discernible already in
pH
4.55.-
4.ssF
~-pv 5.10--
6,00--
Fig. 3. Determination of pl of putative parvalbumins by two-dimensional gel electrophoresis. An extract of an iliofibularis muscle was solubilized in the presence of Ca2+ and separated on a gel in the absence of SDS (see Fig. 1B, lane a). The lane was cut from me gel and placed on top and in the middle of a preformed pH gradient gel (pH 4.0-6.5 (LKB)). The lane is depicted alongside the gel for clarity. Marker proteins with known pl values are visible at beth sides of the gel and a rabbit parvalbumin sample is seen on the far right (PV). Large arrows mark the protein bands at pH 4.90 and 4.95 (component A), 4.90 (component B) and 4.25 (component D). The small arrow indicates two closely spaced bands corresponding to component E.
141 A a
b
B c
161
TABLE I
Properties of parvalbuminsfrom the iliofibularismuscle of X. laevis Apparent relative molecular mass (Mr) was estimated by electrophoresis in the presence of SDS (see Figs. 1 aI~d 2~, and isoelectric point (pl) by isoelectric focusing (see Figs. 3 and 5). These parameters were determined on at least three separate occasions and relative standard deviations were less than 55 for the M r values and about 15 for the p l values.
A--
B--
7 ,,~2/0
Mr pl
!
2 7 0I nm
2 9 I0
Fig. 4. Polyacrylamide gel electrophoresis in the absence of SDS of purified parvalbumins (A) and ultraviolet absorbance spectrum of this preparation (B). Parvalbumin.~ were purified from an iliofibularis muscle as described in Materials and Methods. (A) Samples of the preparation were solubilized in the presence (lane b) or absence of Ca 2+ (lane c) and separated on a gel as described for Fig. lB. Lane a shows a sample of the crude extract from which the parvalbumins were isolated, solubilized in the absence of Ca 2+. (]8) The absorbance spectrum of the parvalbmnin preparation, dissolved in 1~ mM sodium acetate (pH 5.7) at a protein concentration of 5 mg/ml, wa~ recorded using a cuvette with a l-cm light path. The absorbance fel~ ~vithout interruption to a baseline level for wavelengths above 280 ram.
the first dimension in this example) yielded two closely spaced bands around p I 4.5. Rabbit parvalbumin had a pI of 4.90 in agreement with published values [22]. Parvalbumins were then extracted from an iliofibuiaris muscle and purified as described in Materials and Methods. The final preparation was composed for more than 955 of the Mr 14000 and 11000 proteins, the only slight contamination being an Mr 68000 protein. Fig. 4a shows this preparation and the extract from which it was isolated, separated on a gel in the absence of SDS. The three major bands correspond to the components A, B and D tentatively identified above as parvalbumins. The fourth, minor band corresponds to the contaminating Mr 68000 protein. Using this preparation, an ultraviolet spectrum was recorded which showed an absorption profile characteristic of parvalbumins (Fig. 4b). Taken together, these results show that the Mr 14000 and 11000 bands in the crude muscle extract contain a total of four different proteins meeting the criteria for identification as a parvalbumin (Table I). However, the data presented above do not exclude the possible presence of a significant amount of proteins other than parvalbumin in the respective bands, which would preclude the quantification of parvalbumin content of crude
PAl
PA2
PA3
PA4
14000 4.90
11000 4.90
I1000 4.95
11000 4.25
extracts using SDS gels. In order to check whether the Mr 14000 and 11000 bands indeed contain only the acidic proteins identified above, muscle extracts were first separated on an isoelectric focusing gel and then run in the second dimension on an SDS gel. The results in Fig. 5 of a pH 4.0-6.5 gel, show that no proteins other than the identified parvalbumins have an Mr value below 14000, with the possible exception of one acidic protein (pI 4.0), that migrated to an Mr of 11000, and a very small amount of protein visible as a faint spot at M, 14000, p l 4.5. Proteins with M r > 30000 that do not show up on the gel in Fig. 5 had p l values between 7 and 9. This was checked on a pH 3.5-9.5 gel, and also in this pH range we could not detect any proteins of M, < 14 000 (unpublished data). Finally, we checked the possibility that parvalbumin was lost in the pellet that was discarded after extraction pH 4.B5 I
5.10
6.00
I
I
680-
'0
3o0-
(.
12A-
Fig. 5. Two-dimensional gel electrophoresis of a crude muscle extract. A crude extract of an iliofibul~'is muscle was f'wst run on a pH 4.0-6.5 isoelectric focusing gel (LKB) and then in the second dimension in the presence of SDS as described in Materials and Methods. A sample of the crude extract was run on the gel alongside the isoelectric focusing strip. Arrows indicate two unidentified spots at Mr 14000 and 11000. The smear of stain to the right of the parvalbumin spots on this gel is probably caused by diffusion of protein in the interface between the gel strip and the separating gel. This was not observed on other occasions.
B
A m b a
b
c
d
e
f
0
h
c
(1) '0 ×
11-
14-11--
!
C
i t
i
D
<
M r xl[~3 Fig. 6. Polyacrylamidegel electrophoresis using mlnigels. (A) A 15% minigel containing a crude extract of an iliofibularis muscle (lane a), rabbit (lane b) and a sample of the purified parvalbumin preparation (lane c) (see also Fig. 4). (B) A gradient minigel (10-20%) showing the separation of crude extracts of three type 1 fibers (lanes c, d and e) and rabbit parvalbumin (all other lanes). The equivalent of dry weight of ~e fibers that was loaded on the gel was 27/tg for lane c and 19 ttg for lanes d and e. The amounts of rabbit parvalbumin on the gel ranged from 0.25 to 1.25 ltg protein. The position of the M r 14000 and 11000 components is indicated° (C) Absorbance profile of the lower part of lane c ob'a6ned by laser densitometry.Peak areas correspond to 0.45 Itg protein (Mr 14000) and 0.18/tg protein (Mr llO00). The sample in lane d contained 0.24 and 0.08/tg, and the one in lane e contained 0.22 and 0.07 Itg of the Mr 14000 and 11000 components, respectively.
parvalbumin
TABLE II
Parvalbumin content of single fibers of the iliofibularis muscle of X. laevis Sinsle fibers were dissected from freeze-dried iliofibularis muscle and classified according to the criteria in Ref. 27. Fibers were homogenized in solub'dizatiun buffer and crude extracts were separated on minigels (see also Fig. 6). Protein profiles of stained and dried gels were obtained by laser densitometry and peak areas were calculated by computer. Data represent the mean amount (4- S.D.) of Mr 14000 (PAl) and combined Mr 11000 parvalbuming (PA2-4) present in fibers, expressed as mg protein/g fiber wet wt. (fiber wet wt. = 3.73< fiber dry wt.). The number of fibers analyzed is shown in parentheses. Two of the five examined type 2 fibers had undetectable levels of the Mr 11000 isoforms. Differences in the parvalbumin content (total and PAl or PA2-4) between type 1 and type 2 fibers were significant; 2P < 0.005 (Student's t-test).
PAl PA24
TYPE 1
TYPE 2
3.804-0.50 (9) 1.434-0.35 (9)
1.544-0.68 (5) 0.54 4-0.27 (3)
of the soluble fraction. However, extensive solubilization of pellet material followed b y electrophoresis, indicated that no proteins of Mr 14000 or 11000 were present in this fraction.
Quantification of parvalbumin in single muscle fibers Relatively fast (type 1) a n d slow (type 2) contracting and relaxing fibers were isolated from the iliofibularis muscle, a n d extracts were obtained using solub'llization buffer as homogenization medium, as described in Materials a n d Methods. Fig. 6a and b shows examples of a whole muscle extract a n d several single fiber extracts separated o n minigels together with r a b b i t p a r v a l b u m i n a n d the purified parvalbumin p r e p a r a t i o n of Fig. 4. G r a d i e n t gels of 10-20~; acrylamide were initially used, b u t linear 1 5 $ gels yielded similar results and these were routinely used. Fiber d r y weights ranged from 2 0 - 8 0 ltg, and the equivalent of 1 5 - 3 0 Itg was usually enough to obtain a reliable absorbance reading
143 of the parvalbumins from the gel. An example of a laser scan of the low Mr range of a fiber extract is given in Fig. 6c. Table II sums up the average values of the parvalbumin content of type 1 and type 2 fibers. The data show that the Mr 14000 isoform is by far the most abundant in both fiber types and in fact two of the examined type 2 fibers did not yield detectable amounts of the low molecular weight parvalbumins. The total parvalbumin content of the fast type 1 fibers wa~ nearly 3-fold higher than that of the type 2 fibers. Discussion In the present study we examined the possibility of determinin~ the parvalbumin content of single muscle fibers from the ilio~bularis muscle of X. laevis, using protein profiles of crude extracts. Which protein bands after gel electrophoresis correspond to parvalbumin had to be assessed first, because although the Ca2+binding property of parvalbumin has been conserved for over 500 million years of evolution, a large number of isoforms with different physicochemical properties has evolved [1-3]. This polymorphic character of parvalbuwjns is again exemplified by the present results, with four proteins identified in the iliofibularis muscle that met the criteria for parvalbnmin. Our data may be compared to recently published results from a study in which polyclonal antibodies against parvalbumins were used to identify parvalbumins in various muscles of X. laevis [19]..'~u'ee immunoreactive protein bands were f,~und after gel electrophoresis according to Laemmli [2!] at M r 12000, 13 500 and 14000 in sartorius and mylohyoid muscle extracts. Gastrocnemius extracts contained only the 12000 and 14000 species. Given the limited accuracy of M r determinations on gels, our data with one M, 14000 component and one group of M, 11000 components, would seem in good agreement with this latter situation. In Fig. la, the three low M, isoforms comigrated with a frog parvalbumin (unidentified isoform, Sigma). Frog parvalbumins have published M r values ranging from 10000 to 12000 [14-17], and it is likely that the group of proteins correspond to the immunoreactive species at M, 12000 in ReL 19. The major isoform in our study (PAl) co-migrated, as one parvalbn~min species did in Ref. 19, with lysozyme ( M r 14000). This is a relatively high M, value for parvalbumin, but nonetheless, PAl co-migrated with rabbit parvalbumin, which has an M r of 12100 as calculated from the amino acid composition [23]. This obvious overestimation of the M, value may be related to the electrophoresis conditions of Laemmli [21] which were used both by Schwartz and Kay [19] and by us, whereas other methods have been used in all previous studies with rabbit parvalbumin (see for example Ref. 22). Given the particular behavior of rabbit
parvalbumin in this gel system, a true Mr value of around 12000 for the major parvalbumin in X. laevis muscle can not be excluded and is perhaps more likely, since the M r of all parvalbumins thus far characterized does not exceed 12 200. The results have shown that the Mr 14000 and 11000 bands on SDS gels are virtually completely composed of parvalbumins, and that no parvalbumin is lost in the preparation of the extract. The small amount of protein with p I around 4.0 and M r 11000, that was observed once on an isoelectric focusing gel (Fig. 5), may represent a fifth parvalbumin that was not detected in the other analyses. The same may hold true for the acidic protein (pl 4.5) just visible at M, 14000 in Fig. 5. Although the identity of these contaminating proteins is uncertain, their contribution to the parvalbumin peaks" is negli~ole and we eventually chose SDS gels for the quantification. Separation on gels without SDS, although also allowing the separate determination of PA4, was not used, because frequent tailing of the protein bands on such gels prevented accurate measurements. Concerning the absolute parvalbumin content of the fibers, previous studies give a maximal parvalbumin content of frog muscle of 3.5 mg/g wet wt. [14,15,17]. The present results show that due to the heterogeneous composition of the muscle fiber population, higher parvalbumin concentrations can occur in individual cells, which is line with the reported range of parvalbumin contents in different fiber types in mammalian muscle, assessed using immunohistochemical techniques [1,4]o The data in Table II give an average content in type 1 fibers of 5.2 mg/g wet wt., and values as high as 7.0 mg/g wet wt. have been found, which equals 0.59 /tmol/g wet wt., assuming a true Mr of 12 000 for the compone.~ PAl. On the other hand, the lowest parvall-am~ content that we encountered in type 2 fibers was 0.09/~mol/g wet wfL. This broad range of parvalbumin contents, when determined in conjunction with force and heat measurements, as is currently done in our laboratory, should enable a better evaluation of the involvement of parvalbumin in the dynamics and energetics of contracting muscle. With respect to this, the higher parvalbumin content of type 1 as compared to type 2 fibers (Table II) is at least in qualitative agreement with the proposed role of parvalbumin in muscle relaxation [1], since the average initial relaxation rate of the type 1 fibers used in the present study was 33~ higher than that of the type 2 fibers (unpublished obse~ation). Although a thorough characterization of the identified parvalbumins is beyond the scope of this study, some conclusions may be drawn from the obtained data. On the basis of differences in amino acid composition of the isoforms thus far characterized, two genetic lineages, a and fl, have been identified. A higher content of acidic residues in isoforms of fl lineage results in
144 p l values of less than 4.8, whereas more alkaline values are exclusively found in type a parvalbumins [14,15]. Classification of the four parvalbumins found in the iliofibularis on the basis of this criterium, would suggest that three of these are of a lineage (pI > 4.8 [14]): PAl, the most abundant isoform with (apparent) M r 14000 and pI 4.90; PA2, M r 11000 and p l 4.90; and PA3, Mr 11000 and p l 4.95. PA4, the third Mr 11000 isoform, with a p l of 4.25, clearly would be of t-lineage. The identification of this single fl isoform substantiates the already predicted presence of such an isoform on the basis of previous work [18]. In that study, expression of a cDNA sequence encoding a parvalbumin from X. laevis, yielded a protein of Mr 12 000 that showed a striking 76~ homology with carp fl parvalbumin of pI 4.25 [18,28]. Like the carp parvalbumin, this fi isoform lacked tyrosine [18,28], which is present in most fi lineage proteins, but absent in virtually all a isoforms. Because PAl-3 are identified as a isoforms, it is unexpected that we find a strong absorbance around 280 nm characteristic of tyrosine in the ultraviolet spectrum of Fig. 4. Furthermore, when compared with pubfished spectra, the relative peak heights due to phenylalanine (253-268 nm) and tyrosine suggest that the greater portion of the parvalbumin in our preparation contains tyrosine [14,15,17,20]. These data may be reconciled by assuming that at least PAl, the major component in the preparation is closely related to the particular a isoform found in Rana temporaria (pl 4.97) [17] and chicken muscle (pI 4.9) [20]. The amino acid compositions of these proteins is virtually identical and separates them from other a lineage parvalbumins by the presence of one tyrosine residue. The spectrum of Fig. 4 indeed shows a close resemblance to those pubfished for these parvalbumins [17,20]. Comparison of published data on the effect of Ca 2+ binding on the electrophoretic mobility [14,15] and those in Figs. 1 and 4, gives no indication of a correlation between genetic lineage, molecular weight or p I and the extent of this electrophoretic effect. No extra information is therefore obtained from these results, but the striking differences in the magnitude of the effect, which is thought to be related to conformational changes, suggest considerable structural differences between the various isoforms. Given this variation it may at least be concluded that components PA2 and PA3 are very closely related, as they showed identical mobility in the Ca 2+ bound and Mg 2+ bound form. In summary, the described method allows a quantitative analysis of the large differences in total parvalbumin content between individual muscle fibers. Regarding the identification of parvalbumins, our results extend the previously published data on X. laevis muscles [19], by showing that in the iliofibularis muscle the protein fraction of Mr 11000 contains three isoforms of parvalbumin and that the M r 14000 fraction comprises
the major parvalbumin isoform with a probable true Mr of approx. 12 000.
Acknowledgement The excellent technical assistance of Mr. R. Zaremba is gratefully acknowledged.
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