SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth

Organization of actin filaments into a well-organized sarcomere structure is critical for muscle development and function. However, it is not completely understood how sarcomeric actin/thin filaments attain their stereotyped lengths. In an RNAi screen in Drosophila primary muscle cells, we identified a gene, sarcomere length short (sals), which encodes an actin-binding, WH2 domain-containing protein, required for proper sarcomere size. When sals is knocked down by RNAi, primary muscles display thin myofibrils with shortened sarcomeres and increased sarcomere number. Both loss- and gain-of-function analyses indicate that SALS may influence sarcomere lengths by promoting thin-filament lengthening from pointed ends. Furthermore, the complex localization of SALS and other sarcomeric proteins in myofibrils reveals that the full length of thin filaments is achieved in a two-step process, and that SALS is required for the second elongation phase, most likely because it antagonizes the pointed-end capping protein Tropomodulin.     

I-protein is localized at the junctional region of A-bands and I-bands of chicken fresh myofibrils

FITC-labeled antibodies raised against chicken myofibrillar I-protein stained chicken myofibrils, which were fixed with formalin immediately after being cut from the sacrificed chicken breast muscle, at the junctional region of A-bands and I-bands. On the other hand, the antibodies stained the glycerinated myofibrils at the region around Z-bands. Aged glycerinated myofibrils stored in a cold room became stained with the same antibodies at the M-line and the A-band region except for the H-zone and the Z-band. I-Protein, which was originally localized at the A-I junctions, moved to the region around Z-bands and A-bands during the process of preparing myofibrils, paralleling the deterioration of myofibrils. Although I-protein is easily released from its original position, it is not a cytoplasmic protein of muscle but an intrinsic myofibrillar component, because immunoblotting tests showed that I-protein is contained in the myofibrillar fraction and not in the muscular cytoplasmic fraction.     

ULTRASTRUCTURE AND BIREFRINGENCE OF THE ISOLATED MITOTIC APPARATUS OF MARINE EGGS

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.     

STUDIES ON THE STRUCTURE OF MUSCLE : III. PHASE CONTRAST AND ELECTRON MICROSCOPY OF DIPTERAN FLIGHT MUSCLE

1. The flight muscles of blowflies are easily dispersed in appropriate media to form suspensions of myofibrils which are highly suitable for phase contrast observation of the band changes associated with ATP-induced contraction. 2. Fresh myofibrils show a simple band pattern in which the A substance is uniformly distributed throughout the sarcomere, while the pattern characteristic of glycerinated material is identical with that generally regarded as typical of relaxed vertebrate myofibrils (A, I, H, Z, and M bands present). 3. Unrestrained myofibrils of both fresh and glycerinated muscle shorten by not more than about 20 per cent on exposure to ATP. In both cases the A substance migrates during contraction and accumulates in dense bands in the Z region, while material also accumulates in the M region. It is proposed that these dense contraction bands be designated the C_z, and C_m bands respectively. In restrained myofibrils, the I band does not disappear, but the C_z and C_m bands still appear in the presence of ATP. 4. The birefringence of the myofibrils decreases somewhat during contraction, but the shift of A substance does not result in an increase of birefringence in the C_z and C_m bands. It seems therefore that the A substance, if it is oriented parallel with the fibre axis in the relaxed myofibril, must exist in a coiled or folded configuration in the C hands of contracted myofibrils. 5. The fine structure of the flight muscle has been determined from electron microscopic examination of ultrathin sections. The myofibrils are of roughly hexagonal cross-section and consist of a regular single hexagonal array of compound myofilaments the cores of which extend continuously throughout all bands of the sarcomere in all states of contraction or relaxation so far investigated. 6. Each myofilament is joined laterally with its six nearest neighbours by thin filamentous bridges which repeat at regular intervals along the fibre axis and are present in the A, I, and Z, but not in the H or M bands. When stained with PTA, the myofilaments display a compound structure. In the A band, a lightly staining medullary region about 40 A in diameter is surrounded by a densely staining cortex, the over-all diameter of the myofilament being about 120 A. This thick cortex is absent in the I and H bands, but a thinner cortex is often visible. 7. It is suggested that the basic structure is a longitudinally continuous framework of F actin filaments, which are linked periodically by the lateral bridges (possibly tropomyosin). The A substance is free under certain conditions to migrate to the Z bands to form the C_z bands. The material forming the C_m bands possibly represents another component of the A substance. The results do not clearly indicate whether myosin is confined to the A bands or distributed throughout the sarcomere.     

Studies on the structure of muscle. III. Phase contrast and electron microscopy of dipteran flight muscle

1. The flight muscles of blowflies are easily dispersed in appropriate media to form suspensions of myofibrils which are highly suitable for phase contrast observation of the band changes associated with ATP-induced contraction. 2. Fresh myofibrils show a simple band pattern in which the A substance is uniformly distributed throughout the sarcomere, while the pattern characteristic of glycerinated material is identical with that generally regarded as typical of relaxed vertebrate myofibrils (A, I, H, Z, and M bands present). 3. Unrestrained myofibrils of both fresh and glycerinated muscle shorten by not more than about 20 per cent on exposure to ATP. In both cases the A substance migrates during contraction and accumulates in dense bands in the Z region, while material also accumulates in the M region. It is proposed that these dense contraction bands be designated the C(z), and C(m) bands respectively. In restrained myofibrils, the I band does not disappear, but the C(z) and C(m) bands still appear in the presence of ATP. 4. The birefringence of the myofibrils decreases somewhat during contraction, but the shift of A substance does not result in an increase of birefringence in the C(z) and C(m) bands. It seems therefore that the A substance, if it is oriented parallel with the fibre axis in the relaxed myofibril, must exist in a coiled or folded configuration in the C hands of contracted myofibrils. 5. The fine structure of the flight muscle has been determined from electron microscopic examination of ultrathin sections. The myofibrils are of roughly hexagonal cross-section and consist of a regular single hexagonal array of compound myofilaments the cores of which extend continuously throughout all bands of the sarcomere in all states of contraction or relaxation so far investigated. 6. Each myofilament is joined laterally with its six nearest neighbours by thin filamentous bridges which repeat at regular intervals along the fibre axis and are present in the A, I, and Z, but not in the H or M bands. When stained with PTA, the myofilaments display a compound structure. In the A band, a lightly staining medullary region about 40 A in diameter is surrounded by a densely staining cortex, the over-all diameter of the myofilament being about 120 A. This thick cortex is absent in the I and H bands, but a thinner cortex is often visible. 7. It is suggested that the basic structure is a longitudinally continuous framework of F actin filaments, which are linked periodically by the lateral bridges (possibly tropomyosin). The A substance is free under certain conditions to migrate to the Z bands to form the C(z) bands. The material forming the C(m) bands possibly represents another component of the A substance. The results do not clearly indicate whether myosin is confined to the A bands or distributed throughout the sarcomere.     

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.     

Agonist-induced association of tropomyosin with protein kinase Calpha in colonic smooth muscle

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCalpha in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCalpha in the particulate fraction may be due to acetylcholine-induced translocation of PKCalpha to the particulate fraction. To investigate whether the association of PKCalpha with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCalpha indicated direct interaction of tropomyosin with PKCalpha. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCalpha with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.     

Birefringence of Protein Solutions and Biological Systems: II. Studies on TMV, Tropocollagen, and Paramyosin

The intrinsic birefringences of TMV, tropocollagen, and paramyosin were calculated from flow birefringence measurements using the theory of Peterlin and Stuart. The values are -0.029, -0.029, and -0.030, respectively. The intrinsic birefringences of TMV and tropocollagen were measured as a function of the refractive index of the solvent in glycerol-water mixtures. In both cases the values were not constant and became less negative as the refractive index increased. Theoretical calculations showed that the large solvent effect could not be caused by a hydration shell of index different from that of the bulk solvent. It is concluded that either (a) the intrinsic birefringence calculated from the Peterlin-Stuart theory is incorrect or (b) the intrinsic birefringence depends markedly on the solvent. These results are of importance to the problem of quantitative polarized light microscopy since the separation of form and intrinsic birefringence contributions is based on the assumption that intrinsic birefringence is independent of solvent.     

Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. II. Distribution and localization

Antibodies specific for the novel 86 kd protein purified from chicken pectoralis myofibrils stained by indirect immunofluorescence the middle third of each half A-band of isolated myofibrils and myotubes. Pectoralis muscle 86 kd protein, like pectoralis C-protein, displayed a fibre-type specific distribution by being restricted to fast twitch fibres and absent in slow tonic and heart muscle fibres. This was demonstrated by immunoblotting experiments with tissue extracts and by immunofluorescence labelling of cryosections. In primary cell cultures prepared from embryonic chicken breast muscle, 86 kd protein, C-protein and myomesin were all detected in post-mitotic myoblasts where fluorescence was found in a cross-striated pattern along strands of nascent myofibrils. Fluorescence due to the 86 kd protein was restricted to myofibrils within myotubes and no significant labelling of the sarcoplasm was evident. Glycerinated fast twitch muscle fibres, after incubation with antibodies to 86 kd protein, revealed in each half of the A-band nine distinctly labelled stripes, spaced about 43 nm apart. Simultaneous incubation of fibres with antibodies against 86 kd protein and C-protein showed a co-localization of the seven C-protein stripes (stripes 5 to 11), with seven stripes of 86 kd protein. The two additional stripes (stripes 3 and 4) labelled by anti-86 kd antibody continued towards the M-band at the same periodicity from the last C-protein stripe (stripe 5). Thus, partial co-localization of two different thick filament proteins is demonstrated and the identity of transverse stripes at positions 3 and 4 attributed in part to the presence of the new 86 kd protein.     

Birefringence of Protein Solutions and Biological Systems. I

The quantitative interpretation of birefringence of biological structures such as muscle requires a knowledge of intrinsic birefringence of the components. The intrinsic birefringence of fibrous structures as determined by variation of solvent index is positive while the intrinsic birefringence of proteins in solution is negative as calculated by the Peterlin-Stuart theory. As a first step in clarifying this discrepancy the basis of the Peterlin-Stuart theory has been re-examined. The theory has been recalculated from the standpoint of light scattering and extended to particles whose length is not small compared to the wavelength. The birefringence of a system of particles possessing a shell with index different from the bulk solvent has been obtained in order to interpret measurements in mixed solvents.     

Structural change of myofibrils during mitosis of newt embryonic myocardial cells in culture

Newt embryonic myocardial cells can undergo mitosis in culture. The successive changes in the striation pattern of sarcomeres of myofibrils during mitosis were studied by polarization microscopy without fixing or killing the cells. Birefringence of well-organized striation patterns, i.e., bright A-bands and dark I-bands, was clearly visible in interphase cells and did not show any detectable changes during incubation for 3 h or more. Electron microscopy showed the presence of well-organized myofibrils with Z-bands in these interphase cells. When myocardial cells entered the mitotic stage, the birefringence of striation pattern of their myofibrils gradually changed with the pattern in small parts of the myofibrils gradually becoming indistinct (called 'indistinct striation' in this paper). These indistinct regions increased in size during the mitotic stage. In addition, in some regions of the indistinct striation, the birefringence of sarcomeres gradually decreased and finally disappeared (called 'disappearance of sarcomeres' in this paper). No myocardial cells underwent mitosis without these disruptive changes of the myofibril striation patterns. In the post-mitotic stage, the well-organized striation of the myofibrils reappeared. Electron microscopy showed disorganized sarcomeres without Z-bands in the regions of indistinct striation, and no well-organized myofibrils in the regions where the sarcomeres had disappeared. Thus the well-organized myofibrils with Z-bands became transiently disorganized at least in some parts, during mitosis. They were then reorganized into daughter myocardial cells.     

Birefringence measurements of structural inhomogeneities in Rana pipiens rod outer segments.

The birefringence (deltan) of Rana pipiens rod outer segments (ROS) reveals microstructure inhomogeneities not seen with other techniques. In the basal 20-30-micron length of the ROS there is a nearly linear axial gradient in deltan of approximately equal to -2 x 10(-5)/micron. No consistent deltan gradients are found in the distal 20-30 micron of the ROS. Using glycerol imbibition to separate the intrinsic and form birefringence components, we found that the basal deltan gradient was principally due to a gradient of the intrinsic birefringence component. The disk membrane volume fraction decreases uniformly from the basal end to the distal end, while the disk membrane refractive index increases. The contributions of these changes to the form birefringence approximately cancel, so that the form component is fairly uniform along the ROS axis. Because its axial distance from the inner segment is a measure of the time since a disk membrane was formed, these gradients may reflect a disk membrane aging process. Occasionally a highly birefringent, 2-micron-wide band is seen at the basal end ot the ROS, possibly where the envelope membrane folds to form new disk membranes.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

Isolation and characterization of a new 40-kilodalton protein from bovine cardiac muscle

A new protein having a subunit weight of 40,000 has been purified from myosin-extracted bovine cardiac myofibrils. Its amino acid composition and isoelectric point are distinct from actin, eu-actinin, and a variety of sarcoplasmic proteins of similar size. Affinity-purified antibodies made to this protein only react with a single 40-kDa protein band from cardiac myofibrils on immunoblots. The anti-40-kDa protein also shows cross-reactivities with cardiac myofibrils from rabbits, rats, and chickens. Immunofluorescence studies demonstrate that the 40-kDa protein is localized at the Z-bands of cardiac myofibrils and at the intercalated discs. The antibody did not react with skeletal muscle myofibrils by immunofluorescence or immunoblotting. It appears that the 40-kDa protein may play a role in the strong attachments between adjacent myofibrils in cardiac muscle.     

Polarized light microscopy in the study of the molecular structure of collagen and reticulin

Although collagen structure has been studied by polarized light microscopy since the early 19th century and continued since, modern studies and reviews failed to correlate the conclusions based on data obtained by the techniques with those of polarized light microscopy. Collagen I is intensely positively birefringent in respect to length of the fibres; the positive intrinsic birefringence indicates a quasi-crystalline alignment parallel to the fibre and molecule axis of the amino acid residues of the polypeptide chains. This would not have been compatible with a helical structure but has been achieved by similar tilt angles and opposite directions of the coiling and supercoiling. Birefringence characteristics of collagen are also affected by chemical treatments, extractions and staining procedures. Attachment of chemical groups to the anionic charges present on the surface of collagen molecules results in increased positive birefringence in the case of bipolar molecules attached to two or more anionic residues. Unipolar attachment to the same groups, or to the cationic groups of the associated proteoglycans, as well as sulfation or acetylation of hydroxyls of the protein and/or the carbohydrate, reduced or reversed the sign of birefringence. Increased birefringence caused by stretching cannot be due to intramolecular events and is caused by intermolecular changes. The same applies to changes in collagen during aging. Reticulin is a group of different substances which mostly contain collagen III. The pliability and deformability of this collagen is related to its weakly negative birefringence due to large side chains and presence of different and greater amounts of interstitial proteoglycans and other molecules. The so-called reticulin of healing wounds differs in its constitution from other reticulins but is also rich in intermolecular carbohydrate components.     

Caldesmon is a Ca2+-regulatory component of native smooth-muscle thin filaments.

Thin-filament preparations from four smooth muscle types (gizzard, stomach, trachea, aorta) all activate myosin MgATPase activity, are regulated by Ca2+, and contain actin, tropomyosin and a 120000-140000-Mr protein in the molar proportions 1:1/7:1/26. The 120000-140000-Mr protein from all sources is a potent inhibitor of actomyosin ATPase activity. Peptide-mapping and immunological evidence is presented showing that it is identical with caldesmon. Quantitative immunological data suggest that caldesmon is a component of all the thin filaments and that the thin-filament-bound caldesmon accounts for all the caldesmon in intact tissue. The myosin light-chain kinase content of thin-filament preparations was found to be negligible. We propose that caldesmon-based thin-filament Ca2+ regulation is a physiological mechanism in all smooth muscles.     

Intrinsic Birefringence of Poly-γ-Benzyl-l-Glutamate, A Helical Polypeptide, and the Theory of Birefringence

The intrinsic birefringence of macromolecules can be obtained directly from flow birefringence measurements in a solvent whose refractive index matches that of the solute. A small and positive value (approximately 0.01) was found for the helical polypeptide, poly-γ-benzyl-L-glutamate. The birefringence in solvents of varying index calculated from the Peterlin-Stuart theory using this value of the intrinsic birefringence did not agree with experimental values. Considerations of polydispersity and shear deformation indicated that the discrepancy could not be attributed to these effects. Also it could not be explained in terms of specific solvent effects. It is concluded that optical properties cannot be derived from the continuum model employed by Peterlin and Stuart. Much better agreement was obtained with a helical dipole necklace model.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Thin-filament length correlates with fiber type in human skeletal muscle

Force production in skeletal muscle is proportional to the amount of overlap between the thin and thick filaments, which, in turn, depends on their lengths. Both thin- and thick-filament lengths are precisely regulated and uniform within a myofibril. While thick-filament lengths are essentially constant across muscles and species (～1.65 μm), thin-filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that thin-filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified thin-filament lengths that ranged from 1.19 ± 0.08 to 1.37 ± 0.04 μm, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 μm further from the Z-line than the NH(2)-terminus of nebulin in the various biopsies, indicating that human thin filaments have nebulin-free, pointed-end extensions that comprise up to 34% of total thin-filament length. Furthermore, thin-filament length was negatively correlated with the percentage of type 2X myosin heavy chain within the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between thin-filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human thin-filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation.     

Transient electric birefringence of two small DNA restriction fragments of the same molecular weight.

The transient electric birefringence of two small DNA restriction fragments of the same molecular weight, one of which migrates anomalously slowly on polyacrylamide gels, has been investigated. Both fragments exhibit negative birefringence. The decay of the birefringence of the anomalously slowly migrating fragment is 8-9% faster than that of the normally migrating fragment. The faster birefringence decay of the anomalous fragment 12A persists under a variety of buffer conditions, suggesting that it is due primarily to static bending and/or curvature of fragment 12A. In reversing electric fields the absolute amplitude of the birefringence of fragments 12A and 12B decreased about 26% before returning to the steady state value. The minimum in the birefringence occurred faster than expected from the birefringence decay times and decreased with increasing electric field strength, suggesting that the minimum is due to a slow polarization of the ion atmosphere. For both fragments, the rise of the birefringence in the Kerr region is about 10% slower than the field-free decay. The buildup of the negative birefringence is preceded either by an interval when no birefringence is observed or by a small positively birefringent transient, suggesting that a small transverse ionic polarizability is also present. Both DNA fragments exhibit Kerr law behavior over most of the range of electric field strengths investigated. Analysis of the shapes of the saturation curves suggests that differences may exist in the polarization mechanisms of the two fragments.