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.     

Study of the structure of metal-binding centers of calmodulin by EXAFS-spectroscopy

An investigation of Ca2+-binding centers of calmodulin was carried out by EXAFS-spectroscopy. The experimental results for protein preparations of calmodulin in which Ca2+ was isomorphically replaced by Tb3+ were obtained by a spectrometer working at the Institute of Nuclear Physics. For spectra analyses a standard method of Fourier transformation was used. Coincidence main maxima on phi (r) curves and identity of Fourier transformation for calmodulin and parvalbumin in the 2-6 A interval allow to infer the identity of Ca2+-binding centers of calmodulin and parvalbumin.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Study of Ca2+-binding centers of parvalbumin by the distant fine structure of x-ray absorption spectra

An investigation of Ca2+-binding centers of parvalbumin II and III by analysing distant fine structure of X-ray absorption spectra of metal was performed. Protein preparations of parvalbumin II and III in which Ca2+ was isomorphically replaced by Tb3+ were studied. For spectra analyses a standard method of Fourier transformation was used. The middle of the first absorption maximum was taken as origin for energy calculations. Comparison of spectra and modules of Fourier transformations for normalized oscillations of the X-ray spectra of absorption of the II and III components, revealed that the spectra and Fourier-transformants coincide in the 2--6 A interval. This allows to infer the coincidence of the coordinate numbers, average interatomic distances and their dispersions in Ca2+-binding centers of the two protein components.     

Electrospray ionization mass spectrometry: analysis of the Ca2+-binding properties of human recombinant alpha-parvalbumin and nine mutant proteins

A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca2+-binding proteins and other metalloproteins.     

The purification and complete amino acid sequence of the 9000-Mr Ca2+-binding protein from rat placenta. Identity with the vitamin D-dependent intestinal Ca2+-binding protein.

A 9000-Mr Ca2+-binding protein was isolated from rat placenta and purified to homogeneity by h.p.l.c. procedures. The complete amino acid sequence was established for the 78-residue placental protein. A sequence analysis of a minor component of the rat intestinal Ca2+-binding protein (residues 4-78) and a tryptic peptide (residues 55-74), both purified by h.p.l.c., showed both proteins to be identical. Thus this placental 9000-Mr Ca2+-binding protein is the same gene product as the intestinal Ca2+-binding protein whose synthesis is dependent on vitamin D.     

Parvalbumin in non-muscle tissues of the rat. Quantitation and immunohistochemical localization

Parvalbumin, a high affinity Ca2+-binding protein, is known to be expressed only in muscles and brain in the rat. We have investigated its distribution and characteristics in other rat tissues by several biochemical and immunohistochemical methods. Evidence for the presence of parvalbumin in teeth, bone, skin, prostate, seminal vesicles, testes, and ovary is given by two-dimensional polyacrylamide gel electrophoresis, immunoblotting ("Western technique") of one-dimensional gels, and its concentration measured by reverse phase high performance liquid chromatography. The distribution within several parvalbumin-positive organs was monitored by the immunohistochemical peroxidase-antiperoxidase method. In teeth, only ameloblasts reacted with anti-rat parvalbumin serum and in bone the calcified extracellular cartilage was the target of the immunoreaction. The panniculus carnosus was the exclusive site of parvalbumin in the skin. Besides the already known parvalbumin distribution in the brain, parvalbumin is also expressed in distinct cell types of the peripheral nervous system. Leydig cells were found to be the only parvalbumin location in testes. These observations lead us to conclude that parvalbumin in contrast to the multifunctional and constitutive calmodulin must function in Ca2+-dependent processes related to specific cell types.     

Amino acid sequence of alpha chain of sarcoplasmic calcium binding protein obtained from shrimp tail muscle

The isotypes of sarcoplasmic Ca2+ binding protein (SCP) were purified from shrimp tail muscle. SCP exists in a dimeric form. One sample of shrimp contained only alpha A chain, whereas another contained alpha B and beta chains, and a heterodimer of alpha B beta which was not analyzed precisely. The amino acid sequences of the two alpha chains were determined. The two alpha chains are composed of 190 and 192 amino acid residues, respectively. The sequences of the two alpha chains differed in only four amino acids out of 192 residues. The sequences indicate that the alpha chain has three Ca2+-binding sites which are common to EF-hand type Ca2+-binding protein. In the absence of added Ca2+ and Mg2+, the amounts of bound Ca2+ in alpha A, alpha B, and beta chains were 3.0, 3.3, and 2.4 mol/22,000 g protein, respectively. Thus, it is suggested that all three isotypes of shrimp SCP have three Ca2+-binding sites which have high affinity to Ca2+. The sequence homology of shrimp SCP with other EF-hand type Ca2+-binding proteins is very low. The protein having the greatest homology with this SCP was cod parvalbumin; the sequence homology is 18%.     

Interactions of calcium binding proteins, parvalbumin and alpha-lactalbumin, with dipalmitoylphosphatidylcholine vesicles

Interactions of Ca2+ binding proteins, pike (Esox lucius) parvalbumins pI 4.2 and 5.0, and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles were studied by means of scanning microcalorimetry and intrinsic tyrosine and tryptophan fluorescence methods. The interactions of pike parvalbumins are modulated by Ca2+ and Mg2+ binding to the protein and induce some changes in the physical properties of both the proteins and liposomes. Liposomes increased thermal stability of Ca2+-loaded parvalbumin and decreased thermal stability of both Mg2+-loaded and metal-free protein. The interaction of parvalbumin with liposomes affects the phase transition from gel to liquid-crystalline state in liposomes. Ca2+-loaded alpha-lactalbumin interacts with liposomes in its native state while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The results of the microcalorimetric and spectrofluorometric studies are supported by data obtained by means of gel-chromatography on Sepharose 4B. It may be suggested that these metal-modulated interactions of Ca2+-binding proteins with membranes have some functional significance.     

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.     

Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis

A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta-parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos.     

Ca2(+)-binding site of carp parvalbumin recognized by monoclonal antibody.

Monoclonal antibody 235 which was used for immunohistochemical staining of parvalbumin in tissue sections partially protects Lys-54 of carp muscle parvalbumin from reaction with acetic anhydride in the parvalbumin-antibody complex. Lys-54 is located in the CD-loop of parvalbumin and is flanked by the Ca2(+)-ligands Asp-53 and Ser-55 of the Ca2(+)-site I. Another monoclonal antibody against carp parvalbumin, mca 239, partially protects lysine residues 27, 32, 87 and 107, indicating that this antibody is directed against a discontinuous epitope distant from the two Ca2(+)-binding sites of parvalbumin.     

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.     

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.     

The effect of disodium ethane-1-hydroxy-1,1-diphosphonate on the metabolism of calcitriol in chicks.

Decreased intestinal absorption of Ca2+ occurs in response to treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The effect is due to decreased 1-hydroxylation of calcidiol (25-hydroxycholecalciferol) in the kidney. In an attempt to establish whether impairment of vitamin D metabolism at steps beyond kidney hydroxylation occurs due to treatment with EHDP, chicks were depleted of vitamin D and were treated with calcitriol (1,25-dihydroxycholecalciferol) as their sole source of the vitamin. The chicks were then divided into two groups, one being treated with EHDP while the second group served as control. Intestinal absorption of Ca2+ in the EHDP-treated group was found to be impaired, along with decreases in concentrations of calbindin D28K (the 28,000-Mr vitamin D-dependent Ca2+-binding protein). When the chicks were dosed with [3H]calcitriol, significantly lower concentrations of the sterol were detected in the duodena of EHDP-treated birds. Measurement of levels of receptors for calcitriol in duodena showed no difference between groups, but levels of calcitriol in sera were considerably lower in the EHDP-treated group along with the elevated biliary and urinary excretion of glucuronidated conjugates. It is therefore concluded that treatment with EHDP results in increased catabolism of calcitriol in addition to the known suppression of the renal production of the hormone.     

Binding shift assay of parvalbumin, calmodulin and carbonic anhydrase by high-performance capillary electrophoresis

Shifts in mobility caused by binding of Ca2+ to calmodulin and parvalbumin were studied using high-performance capillary electrophoresis in a Tris-glycine buffer, rather than conventional polyacrylamide gel electrophoresis which requires larger amounts of sample and longer assay time. A Zn(2+)-binding protein, carbonic anhydrase, also showed a partial shift in mobility following Zn(2+)-binding.     

Altered synthesis of the 26-kDa heat stress protein family and thermotolerance in cell lines with elevated levels of calcium-binding proteins

Using a bovine papilloma virus-based vector, mouse mammary adenocarcinoma cells have been transformed to express elevated amounts of functional calmodulin (CaM) (Rasmussen and Means, 1987) and another Ca2(+)-binding protein, parvalbumin (PV) (Rasmussen and Means, 1989) that is not normally synthesized in these cells. Parental cells (C127) and cells transformed by the vector alone (BPV-1), the vector containing a CaM gene (CM-1), or the vector containing parvalbumin (PV-1) were used to study the effect of increased synthesis of Ca2(+)-binding proteins on heat-stress protein (HSP) synthesis and cell survival following heating at 43 degrees C. The induction, stability, and repression of the synthesis of most HSPs after 43 degrees C heating was not significantly affected by increased amounts of Ca2(+)-binding proteins, but the rate of synthesis of all three isoforms of the 26-kDa HSP (HSP26) was greatly reduced. C127 cells, which have about one half as much CaM as do BPV-1 cells, synthesized the most HSP26. CM-1 cells, which have more than fourfold higher levels of CaM than do BPV-1 cells, had a rate of synthesis of HSP26 approaching that of unheated cells. BPV-1 cells, with a two-fold increase in CaM, were intermediate in HSP26 synthesis. This effect on HSP26 synthesis may be largely related to the Ca2(+)-binding capacity of CaM rather than to a specific CaM-regulated function, since PV-1 cells also showed reduced rates of HSP26 synthesis. Survival experiments showed that reduced HSP26 synthesis in cells with increased amounts of Ca2(+)-binding proteins did not significantly alter intrinsic resistance to continuous 43 degrees C heating. Thermotolerance was not reduced and appeared to develop more rapidly in CM-1 and PV-1 cells. These results suggest that (1) the signal for HSP26 synthesis can be largely abrogated by elevated Ca2+ binding protein levels, and (2) if these HSPs are involved in thermotolerance development, that function may be associated with intracellular Ca2+ homeostasis.     

Effect of calcium binding on the quenching time of self-fluorescence of Ca-binding proteins

Lifetimes of phenylalanine, tyrosine and tryptophan self-fluorescence of three Ca2+-binding proteins (parvalbumins pI 4.47 and 3.95 and bovine alpha-lactalbumin) in the Ca2+-saturated state and without Ca2+ were measured on a device functioning in a channel of synchrotron radiation of the Lebedev Physical Institute electron accelerator C-60 with a single photon counting system. The decay curve of phenylalanine fluorescence of Ca2+-saturated parvalbumin pI 4.47 is two-exponential, which results from the presence of two subsystems of phenylalanine residues in this protein. Radiation of these subsystems is almost independent of one another. Detachment of Ca2+ from protein disturbs these subsystems. In case of tyrosine fluorescence of carp parvalbumin pI 3.95 a change in the quantum yield value of the stationary fluorescence induced by elimination of Ca2+ proceeds without a change of fluorescence lifetime. This seems to be related to the existence of static quenching of fluorescence in this case at the expense of complex formation between the chromophore and some adjacent quenching groups. Detachment of Ca2+ from alpha-lactalbumin induces conformational changes in its structure. The latter result in a transition of a number of tryptophane residues from its interior to the surface of the globule which is reflected in an increase of fluorescence quantum yield duration. It is concluded that in Ca2+-saturated alpha-lactalbumin some tryptophane residues are located near the quenching groups (dynamic quenching), most likely the disulfide bridges.     

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.