Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis

We have used immunocytological techniques to examine the developmental expression of the Ca2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells.     

Purification and characterization of the parvalbumin from monkey skeletal muscle

1. A high affinity Ca2+ binding and low mol. wt protein, parvalbumin, was purified from monkey skeletal muscle. 2. As compared with other animals, only one component and a lower content of monkey parvalbumin were found. 3. This may suggest that both the component and the content of parvalbumin decreases with biological evolution. 4. The parvalbumin was found to have a mol. wt of 11,400, a pI of 5.1, a high aspartic acid and lysine content, maximum absorption at around 260 nm, a blocked amino-terminal, an immunological distinction, 2 mol Ca2+ binding/mol, and a conformational change by Ca2+ binding. 5. Parvalbumin was shown to have alpha type properties.     

Fluorescence studies of the conformational dynamics of parvalbumin in solution: lifetime and rotational motions of the single tryptophan residue

The fluorescence properties of the single tryptophan residue in whiting parvalbumin were used to probe the dynamics of the protein matrix. Ca2+ binding caused a blue-shift in the emission (from lambda max = 339 to 315 nm) and a 2.5-fold increase in quantum yield. The fluorescence decay was nonexponential in both Ca2(+)-free and Ca2(+)-bound parvalbumin and was best described by Lorentzian lifetime distributions centered around two components: a major long-lived component at 2-5 ns and a small subnanosecond component. Raising the temperature from 8 to 45 degrees C resulted in a decrease in both the center (average) and width (dispersion) of the major lifetime distribution component, whereas the center, width, and fractional intensity of the fast component increased with temperature. Arrhenius activation energies of 1.3 and 0.3 kcal/mol were obtained in the absence and in the presence of Ca2+, respectively, from the temperature dependence of the center of the major lifetime distribution component. Direct anisotropy decay measurements of local tryptophan rotations yielded an activation energy of 2.3 kcal/mol in Ca2(+)-depleted parvalbumin and indicated a correlation between rotational rates and lifetime distribution parameters (center and width). Ca2+ binding produced a decrease in the width of the major lifetime distribution component and a decrease in tryptophan rotational mobility within the protein. There was a rough correlation between these two parameters with changes in Ca2+ and temperature, so that both measurements may be taken to indicate that the structure of Ca2(+)-bound parvalbumin was more rigid than in Ca2(+)-depleted parvalbumin.     

Expression of the Lewis group carbohydrate antigens during Xenopus development

We have examined the pattern of expression of the Lewis group carbohydrate antigens during the development of African toad Xenopus laevis. One of these antigens, Lewis x (Le(x), also known as SSEA-1), was previously shown to be involved in cell-cell adhesion in early mouse embryos and teratocarcinoma stem cells. Recently another member of these antigens, sialyl-Le(x), was found to be one of the major ligands for the selectin family of cell-cell adhesion molecules. In order to study the role of carbohydrate-mediated cell adhesion during Xenopus development, we first studied the expression pattern of the Le(x). We found that Le(x)was not expressed in early embryos, started to be expressed at the tail bud stage in anterior regions of the body such as the cement gland or head skin, and was gradually showed more posterial expression at later stages. At tadpole stage, it was also expressed on specific cell bodies in brain, and in axon region in brain and neural retina. Antibodies against Le(x)blocked neurite outgrowth in the explant culture of tadpole brain. One of the candidates for Le(x)carrier protein in the tadpole brain is a 200 kDa glycoprotein detected by Western blotting. In adult tissues, it was expressed in brain, testis, and gut, but not in kidney, lung, spleen, ovary, or muscle. We also examined the expression patterns of other Lewis group antigens. Among them, sialyl-Le(x)was expressed on endothelial cells and on leukocytes, suggesting the possibility that it functions as a ligand for selectin in Xenopus.     

Neural control of gene expression in skeletal muscle. Calcium-sequestering proteins in developing and chronically stimulated rabbit skeletal muscles.

Tissue contents of the sarcoplasmic-reticulum Ca2+-ATPase (Ca2+ +Mg2+-dependent ATPase), of calsequestrin and of parvalbumin were immunochemically quantified in homogenates of fast- and slow-twitch muscles of embryonic, maturing and adult rabbits. Unlike parvalbumin, Ca2+-ATPase and calsequestrin were expressed in embryonic muscles. Presumptive fast-twitch muscles displayed higher contents of these two proteins than did presumptive slow-twitch muscles. Calsequestrin steeply increased before birth and reached adult values in the two muscle types 4 days after birth. The main increase in Ca2+-ATPase occurred during the first 2 weeks after birth. Denervation of postnatal fast- and slow-twitch muscles decreased calsequestrin to amounts typical of embryonic muscle and suppressed further increases of Ca2+-ATPase. Denervation caused slight decreases in Ca2+-ATPase in adult fast-twitch, but not in slow-twitch, muscles, whereas calsequestrin was greatly decreased in both. Chronic low-frequency stimulation induced a rapid decrease in parvalbumin in fast-twitch muscle, which was preceded by a drastic decrease in the amount of its polyadenylated RNA translatable in vitro. Tissue amounts of Ca2+-ATPase and calsequestrin were essentially unaltered up to periods of 52 days stimulation. These results indicate that in fast- and slow-twitch muscles different basal amounts of Ca2+-ATPase and calsequestrin are expressed independent of innervation, but that neuromuscular activity has a modulatory effect. Conversely, the expression of parvalbumin is greatly enhanced by phasic, and drastically decreased by tonic, motor-neuron activity.     

Preparation and characterization of the major isotype of parvalbumin from skeletal muscle of the toad (Bufo bufo japonicus)

The major isotype of parvalbumin has been isolated from the skeletal muscle of the toad, Bufo bufo japonicus. Unlike the skeletal muscle of every frog so far examined (Rana esculenta, Rana temporaria, and Rana catesbeiana), which contains two major isotypes of parvalbumins, toad skeletal muscle has been shown to contain only one isotype, but the content of parvalbumin in toad skeletal muscle was similar to the sum of those of the two isotypes in skeletal muscles of frogs. This feature of toad skeletal muscle is advantageous to clarify the physiological role of parvalbumin. The relative molecular mass of toad parvalbumin was estimated to be 12,200 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 4.81 by polyacrylamide gel isoelectric focusing. The amino acid composition indicated that toad parvalbumin corresponds to bullfrog (R. catesbeiana) pI 4.97 parvalbumin, showing that toad parvalbumin is genetically an alpha-parvalbumin. It was also revealed by the amino acid composition that toad parvalbumin is distinctly different from any of the parvalbumins from frogs. The ultraviolet spectrum of toad parvalbumin is consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca2+-loaded form vs. the metal-free form indicates that some Phe residues in the toad parvalbumin molecule are affected by a conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg2+-loaded forms of toad parvalbumin migrated twice as fast as the Ca2+-loaded form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chicken parvalbumin. Comparison with parvalbumin-like protein and three other components (Mr = 8,000 to 13,000).

Procedures for a rapid isolation and purification of parvalbumin (Mr = 12,600), parvalbumin-like protein (Mr = 12,800), and three other polypeptides with molecular weights of 12,400 (Component 1), 11,700 (Component 2), and 8,000, respectively, from chicken leg muscle, are described. A direct comparison of parvalbumin with these other proteins showed distinct differences in the amino acid compositions, charge, and immunological behavior. Parvalbumin has two high affinity sites for Ca2+ with a KDiss less than or equal to 10(-6) M (Blum, H. E., Lehky, P., Kohler, L., Stein, E.A., and Fischer, E. H. (1977) J. Biol. Chem. 252, 2834-2838), in contrast to parvalbumin-like protein. Components 1 and 2, and the Mr = 8,000 protein, where only low affinity sites for Ca2+ could be detected (KDiss greater than 10(-3) M). From our results it is concluded that the co-extracted proteins do not constitute isoproteins of parvalbumin. The very low affinity for Ca2+ suggests that these proteins are not involved in processes of Ca2+ transport or Ca2+ regulation as proposed for parvalbumin. Parvalbumin could not be localized within isolated myofibrils and also did not accumulate in primary myogenic cell cultures together with proteins forming the myofibrillar structure. Parvalbumin was not even detected in myotubes in which myofibrils and sarcoplasmatic reticulum were already assembled and functioning. Parvalbumin (or cross-reacting material) was detected in leg muscle and brain 1 day after hatching of the chick. Possible roles for parvalbumin are discussed.     

Spatio-temporal distribution of the protein of Xenopus vasa homologue (Xenopus vasa-like gene 1, XVLG1) in embryos

In order to know when the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila , the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.     

Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells

In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.     

Ca2+-binding parvalbumin in rat testis. Characterization, localization, and expression during development

Parvalbumin, a Ca2+-binding protein, was isolated from rat testis. This is the first demonstration of the protein in endocrine glands. By using a rat parvalbumin cDNA probe, parvalbumin mRNA was demonstrated in the testis, indicating that the protein is synthesized in this tissue and that testis parvalbumin is a product of the same gene as the one encoding for muscle parvalbumin. Parvalbumin was localized by immunohistochemical methods in the Leydig cells and in the acrosome region of maturing spermatids (stages 1-15). The expression of parvalbumin during testis development was followed. High parvalbumin protein and mRNA levels were found at stages of highest Leydig cell activity, i.e. at late fetal stages until birth and again around postnatal day 50. This suggests that parvalbumin may be involved in the production of testosterone in Leydig cells, a process which is highly dependent on calcium.     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis

The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) 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 parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. 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 minigels, yielded mean values (mg protein/g wet wt., +/- 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 Mr 14,000 isoform relative to the combined Mr 11,000 isoforms.     

Role of calcium ions in the control of embryogenesis of Xenopus. Changes in the subcellular distribution of calcium in early cleavage embryos after treatment with the ionophore A23187.

Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Parvalbumin in rat kidney. Purification and localization

The Ca2+-binding parvalbumin has been purified for the first time from rat kidney. Its biochemical and immunological properties were indistinguishable from the muscle counterpart. By immunohistochemical methods parvalbumin was localized in part of the distal tubule and proximal collecting duct, similar to the vitamin D-dependent Ca2+-binding protein, calbindin-28K. Parvalbumin was found to be independent of the vitamin D status of the animal since its concentration remained unchanged in kidney extracts of normal, rachitic and vitamin D-replete rats. Both proteins may be involved in the regulation of intracellular Ca2+ in kidney.     

The time-course of Ca2+ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca2+.

We have modeled the time-course of Ca2+ binding to calmodulin, troponin, parvalbumin, and myosin in response to trains of transient increases in the free myoplasmic calcium ion concentration (pCa). A simple mathematical expression was used to describe each pCa transient, the shape and duration of which is qualitatively similar to those thought to occur in vivo. These calculations assumed that all individual metal binding sites are noninteracting and that Ca2+ bind competitively to the Ca2+-Mg2+ sites of troponin, parvalbumin, and myosin. All the on-and-off rate constants for both Ca2+ and Mg2+ were obtained either from the literature or from our own research. The percent saturation of the Ca2+-Mg2+ sites with Ca2+ was found to change very little in response to each pCa transient in the presence of 2.5 X 10(-3)M Mg2+. Our analysis suggests that the Ca2+ content of these sites is a measure of the intensity and frequency of recent muscle activity because large changes in the Ca2+ occupancy of these sites can occur with repeated stimulation. In contrast, large rapid changes in the amount of Ca2+ bound to the Ca2+-specific sites of troponin and calmodulin are induced by each pCa transient. Thus, only sites of the "Ca2+-specific" type can act as rapid Ca2+-regulatory sites in muscle. Fluctuation in the total amount of Ca2+ bound to these sites in response to various types of pCa transients further suggests that in vivo only about one-half to one-third of the total steady-state myofibrillar Ca2+-binding capacity exchanges Ca2+ during any single transient.