Caldesmon from rabbit liver: molecular weight and length by analytical ultracentrifugation

Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 +/- 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 +/- 0.02 S. yields an apparent length of 53 +/- 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-6098).     

Localization and characterization of a 7.3-kDa region of caldesmon which reversibly inhibits actomyosin ATPase activity.

Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca(2+)-calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S-1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin.     

Turkey gizzard caldesmon: molecular weight determination and calmodulin binding studies

Sedimentation equilibrium and sedimentation velocity measurements demonstrate that turkey gizzard caldesmon is an elongated molecule of molecular mass 75 +/- 2 kDa. The frictional ratio (2.14) is consistent with a prolate ellipsoid of axial ratio 24, corresponding to an apparent length and width of 516 and 21.5 A, respectively. As was previously determined for chicken gizzard caldesmon [Graceffa, P., Wang, C.-L.A., & Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202], this molecular weight is appreciably smaller than the value (approximately 135,000) estimated from the results of NaDodSO4 gel electrophoresis experiments. However, a significant difference between the true molecular weights of turkey and chicken gizzard caldesmons--75,000 versus 93,000--also points to probable molecular weight variations within the subclass. Binding measurements, based on perturbation of the intrinsic tryptophan fluorescence of caldesmon in the presence of calmodulin, show that the interaction between the two proteins is strongly ionic strength and temperature dependent. Dissociation constants of 0.075 and 0.38 microM were determined in solutions containing 0.1 and 0.2 M KCl, respectively, at 24.3 degrees C. Fluorescence emission spectra and fluorescence anisotropy excitation spectra indicate that the tryptophanyl residues of caldesmon are located in solvent-accessible regions of the molecule, where they exhibit a high degree of mobility even when calmodulin is bound.     

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.     

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.     

Polypeptide molecular weights of the (Na+,K+)-ATPase from porcine kidney medulla

The molecular weights of the polypeptide chains from (Na+,K+)-ATPase of porcine kidney medulla have been determined by analytical sedimentation equilibrium. The alpha-subunit molecular weight is 93 900, and the beta subunit is a glycoprotein with a polypeptide molecular weight of 32 300 (41 400 including protein and carbohydrate). Amino acid and carbohydrate compositions are presented together with related properties (i.e., partial specific volumes, extinction coefficients, and hydrophobic/hydrophilic amino acid content).     

Identification of a 15 kilodalton actin binding region on gizzard caldesmon probed by chemical cross-linking

Fluorescent labeling, limited proteolysis, amino acid sequence determinations, affinity chromatography and specific chemical crosslinking were used to determine the smallest fragment of gizzard caldesmon that interacts with actin. The time course of cleavage with thrombin or submaxillaris arginase-C protease indicates that 90kDa and 35kDa fragments are the two major pieces of the 120kDa native protein. Amino acid sequence determination indicates that the 90kDa fragment is the N-terminal portion of the molecule. Further degradation gave rise to a 15kDa product whose N-terminal amino acid sequence was determined within the first 28 amino acids. Carbodiimide crosslinking with actin revealed that the 15kDa part of the molecule is probably not involved in the actin binding process but may participate in a twisting of the F-actin filament and be responsible of the caldesmon regulatory function during smooth muscle contraction.     

Molecular weight,amino acid composition and other properties of membrane-bound ATPase fromBacillus megaterium KM

The Ca²⁺-activated ATPase fromBacillus megaterium KM has a molecular weight of 379000 as determined by sedimentation equilibrium in the analytical ultracentrifuge and 410000 as determined from sedimentation and diffusion coefficients. These values compare closely with molecular weights estimated for similar ATPases fromStreptococcus faecalis and mitochondria. On polyacrylamide gel electrophoresis in sodium dodecylsulfate two classes of subunit of molecular weight 68000 and 65000 are seen. They seem to be present in approximately equal proportion. The amino acid analysis gives a minimum molecular weight of 6250 and the amino acid composition is extremely close to that ofS. faecalis. The enzyme is denatured at 55°C and insensitive to oligomycin or ruthenium red in either the membrane-bound or soluble forms. The ATPase is estimated to comprise approximately 1% of the total cytoplasmic membrane protein.     

Scattering correction to the absorbance, wavelength dependence of the refractive index increment, and molecular weight of the bovine liver glutamate dehydrogenase oligomer and subunits

The wavelength dependence of the refractive index increments of bovine serum albumin and bovine liver glutamate dehydrogenase solutions was determined in the range 650–300 nm. It was shown, by measuring the extinction coefficients of glutamate dehydrogenase under conditions of widely differing molecular weights, that a significant scattering correction need not be applied to correct extinction measurements in the absorbtion band. The molecular weight of glutamate dehydrogenase oligomer determined by light scattering and sedimentation equilibrium agrees with the value calculated from the primary structure, if a recently reported value of the extinction coefficient is used.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

Microinjection of nonmuscle and smooth muscle caldesmon into fibroblasts and muscle cells

Caldesmon is present in a high molecular mass form in smooth muscle and predominantly in a low molecular mass form in nonmuscle cells. Their biochemical properties are very similar. To examine whether these two forms of caldesmon behave differently in cultured cells, we microinjected fluorescently labeled smooth muscle and nonmuscle caldesmons into fibroblasts. Simultaneous injection of both caldesmons into the same cells has revealed that both high and low relative molecular mass caldesmons are quickly (within 10 min) and stably (over 3 d) incorporated into the same structures of microfilaments including stress fibers and membrane ruffles, suggesting that nonmuscle cells do not distinguish nonmuscle caldesmon from smooth muscle caldesmon. The effect of calmodulin on the incorporation of caldesmon has been examined by coinjection of caldesmon with calmodulin. We have found that calmodulin retards the incorporation of caldesmon into stress fibers for a short period (10 min) but not for a longer incubation (30 min). The behavior of caldesmon in developing muscle cells was also examined because we previously observed that caldesmon disappears during myogenesis (Yamashiro, S., R. Ishikawa, and F. Matsumura. 1988. Protoplasma Suppl. 2: 9-21). We have found that, in contrast to its stable incorporation into stress fibers of fibroblasts, caldesmon is unable to be incorporated into thin filament structure (I-band) of differentiated muscle.     

Immunoreactive forms of caldesmon in cultivated human vascular smooth muscle cells

150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.     

Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

Solutions of crystalline beta-lactamase I and beta-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for beta-lactamase I and 35600 for beta-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.     

Purification of caldesmon and myosin light chain (MLC) kinase from arterial smooth muscle: comparisons with gizzard caldesmon and MLC kinase

We have developed a simple and conventional purification method for caldesmon and MLC kinase from bovine arterial smooth muscle, and compared the arterial and gizzard proteins. Arterial caldesmon shares the alternative binding to calmodulin or F-actin in a Ca2+-dependent manner and the antigenic determinants with the gizzard protein. Both caldesmons have the same association constant with F-actin (1.3-1.7 X 10(7) M-1) and the same maximum binding (1 caldesmon per 12-14 actins). However, the molecular weight of arterial caldesmon (dimer of a 148 kDa polypeptides) was slightly different from that of gizzard caldesmon (heterodimer of 150/147 kDa polypeptides). The molecular weight of arterial MLC kinase (160 kDa) was much larger than that of the gizzard enzyme (135 kDa). The enzyme activities of both MLC kinases were comparable (Km = 9.5 microM, Vmax = 12.5 mumol/min X mg). The association constant of the arterial enzyme to F-actin (5.1 X 10(6) M-1) was much larger than that of the gizzard enzyme (9.0 X 10(5) M-1) but the maximum binding was the same (1 enzyme per 12-13 actins). Immunocytochemical examinations showed that caldesmon and MLC kinase in cultured arterial cells have a restricted localization along the stress fibers, suggesting functional linkages between both proteins and actin filaments in vivo.     

Physical properties of L-asparaginase from Serratia marcescens.

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.     

Beta-Adrenergic receptors in brown-adipose tissue. Characterization and alterations during acclimation of rats to cold.

Aldehyde dehydrogenase from bovine liver has been purified to homogeneity. Amino acid composition showed a high content of cysteine of 32 mol/mol enzyme. The enzyme is composed of four identical subunits as judged by sodium dodecyl sulfate gel electrophoresis and end-group analysis. The molecular weight was determined to be 220 000 +/- 10 000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD+, glyceraldehyde and acetaldehyde were found to be 47 micron, 170 micron and 130 micron, respectively.     

Sedimentation behavior of native and reduced apolipoprotein A-II from human high density lipoproteins.

The solution properties of human serum apolipoprotein A-II, both in the native and in the reduced forms, were investigated by the technique of sedimentation equilibrium in the analytical ultracentrifuge. For both proteins, the apparent weight average molecular weights determined in neutral buffer systems were found to be dependent on protein concentration and invariant with the rotor speeds used (16,000 to 44,000 rpm) indicating a reversible self-association. These results were also found to be independent of temperature between 5 and 30 degrees C. The pattern of self-association of native apolipoprotein A-II could best be described by a monomer-dimer-trimer equilibrium, in agreement with previously reported data (Vitello, L B., and Scanu, A. M. (1975), Biochemistry 15, 1161). The self-association pattern of apolipoprotein A-II reduced in the presence of 50 mM dithiothreitol conformed with a monomer-dimer-tetramer equilibrium similar to that reported for the native single chain apolipoprotein A-II of the rhesus monkey (Barbeau, D. L., et al. (1977), J. Biol. Chem. 252, 6745), but differing significantly from that reported for the reduced and carboxymethylated human product (Osborne, J. C. , et al. (1975), Biochemistry 14, 3741).     

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.     

A parameterized overspeeding method for the rapid attainment of low-speed sedimentation equilibrium

The approach of a solution of dilute, monodisperse, globular macromolecules to low-speed sedimentation equilibrium in an ultracentrifuge is simulated by numerical integration of the Lamm equation. Various combinations of overspeed time and angular velocity are used to assess the conditions needed to minimize the time it takes the solution to attain sedimentation equilibrium. The optimal overspeeding time and angular velocity are determined over a wide range of values of the molecular weight (relative molar mass) of the solute and the radial distance between the meniscus and base of the solution. The results may be expressed as simple functions which allow facile calculation of (a) the optimal overspeeding time and velocity, and (b) the time required to reach sedimentation equilibrium. The results are in reasonable agreement with previous analytical solutions which were based on several simplifying assumptions. The parameterized overspeeding procedure is shown to be robust over a wide range of conditions, and typically leads to a greater than 5-fold reduction in centrifugation time.     

Spectrofluorimetric studies on C-terminal 34 kDa fragment of caldesmon

Analysis of the tryptophan fluorescence emission spectra of caldesmon and its 34 kDa C-terminal fragment indicates that all tryptophan residues are located on the surface of the molecule, accessible to solvent. All three tryptophan residues of the 34 kDa fragment and four of the five tryptophan residues of intact protein are accessible to free water, whereas one located in the N-terminal region of molecule is accessible only to bound water molecules. The temperature dependence of the fluorescence parameters indicates higher thermal stability of the 34 kDa fragment than the whole caldesmon molecule. The interaction of the 34 kDa fragment of caldesmon (like that of the intact molecule) with calmodulin is accompanied by a blue shift of the fluorescence emission maximum and an increase in the relative quantum yield. Computer-calculated binding constants show that the binding of calmodulin to the 34 kDa fragment (K = 2.5 x 10(5) M-1) is of two orders of magnitude weaker than that to intact caldesmon (K = 1.4 x 10(7) M-1). The interaction with tropomyosin results in a blue shift of the spectrum of the 34 kDa fragment, yet there is no effect on the spectrum of intact caldesmon. Binding constants of tropomyosin to caldesmon (K = 3.8 x 10(5) M-1) and its 34 kDa fragment (K = 2.3 x 10(5) M-1) are similar. Binding of calmodulin to caldesmon and to the 34 kDa fragment affects their interaction with tropomyosin.