Calcium-binding Protein Content Research Articles

Temporal lobe epilepsy with and without psychosis: exploration of hippocampal pathology including that in subpopulations of neurons defined by their content of immunoreactive calcium-binding proteins.

We have investigated relationships between hippocampal/temporal lobe neuropathology and psychosis in subjects with temporal lobe epilepsy, paying particular attention to possible differences in density of hippocampal neurons immunoreactive for calcium-binding proteins. There was a trend for a greater prevalence of left handedness in the psychotic (n = 6) than the non-psychotic (n = 26) cases (P = 0.0504). Psychotic cases also differed from non-psychotic ones in having: (1) more focal lesions outside the hippocampus (P = 0.006); (2) less severe CAI neuron loss (P = 0.015); and (3) a trend, after Bonferroni correction, for a higher density of calbindin-immunoreactive neurons in the CA4 (P = 0.022). An additional finding was that dentate granule cell dispersion was significantly associated with the presence of a reduced density of calretinin-immunoreactive neurons in CA4 (P = 0.002) and with a more severe loss of CA4 neurons visible with Nissl stain (P = 0.003). Thus, cases of temporal lobe epilepsy with psychosis were distinguishable on the basis of a higher density of calbindin-reactive neurons in CA4 as well as on more general aspects of their pathology.

Distribution of neurons immunoreactive for calcium-binding proteins varies across areas of cat primary somatosensory cortex

The primary somatosensory (SI) cortex in the cat contains four cytoarchitectonic areas that appear to contain separate body representations and have different functions. We tested whether functional differences among these areas are reflected in the densities of neurons containing each of three calcium-binding proteins: parvalbumin (PV), calbindin (CB), and calretinin (CR). Colocalization experiments revealed that CR was localized in a population of neurons distinct from those containing PV or CB. The general laminar distributions of the three calcium-binding proteins were similar to those described in other species and cortical areas, but there were significant density differences in layers II and III across SI. The density of PV-immunoreactive neurons was higher in areas 3b and 1 than in areas 3a and 2. CB-immunoreactive neurons were found in higher densities in anterior SI than in posterior SI, and the pattern of CR-immunoreactive neurons was reciprocal to that of CB, with significantly higher densities in posterior regions of SI. Since the firing characteristics of nonpyramidal neurons appear to be related to their calcium-binding protein content, differences in regional distributions of these neurons in layers II and III may contribute to functional differences between the cytoarchitectonic areas of SI cortex.

On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins.

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.

The Python pit organ: imaging and immunocytochemical analysis of an extremely sensitive natural infrared detector

The Python infrared-sensitive pit organ is a natural infrared imager that combines high sensitivity, ambient temperature function, microscopic dimensions, and self-repair. We are investigating the spectral sensitivity and signal transduction process in snake infrared-sensitive neurons, neither of which is understood. For example, it is unknown whether infrared receptor neurons function on a thermal or a photic mechanism. We imaged pit organs in living Python molurus and Python regius using infrared-sensitive digital video cameras. Pit organs were significantly more absorptive and/or emissive than surrounding tissues in both 3–5 μm and 8–12 μm wavelength ranges. Pit organs exhibited greater absorption/emissivity in the 8–12 μm range than in the 3–5 μm range. To directly test the relationship between photoreceptors and pit organ infrared-sensitive neurons, we performed immunocytochemistry using antisera directed against retinal photoreceptor opsins. Retinal photoreceptors were labeled with antisera specific for retinal opsins, but these antisera failed to label terminals of infrared-sensitive neurons in the pit organ. Infrared-receptive neurons were also distinguished from retinal photoreceptors on the basis of their calcium-binding protein content. These results indicate that the pit organ absorbs infrared radiation in two major atmospheric transmission windows, one of which (8–12 μm) matches emission of targeted prey, and that infrared receptors are biochemically distinct from retinal photoreceptors. These results also provide the first identification of prospective biochemical components of infrared signal transduction in pit organ receptor neurons.

Axonal expression sites of tyrosine hydroxylase, calretinin- and calbindin-immunoreactivity in striato-pallidal and septal nuclei of the rat brain: a double-immunolabelling study

Besides the dopaminergic afferent projection system, calbindin (CALB)- and calretinin (CR)-immunoreactive fibres of intrinsic and extrinsic origin represent the most abundant axonal categories in the rat striatal and lateral septal areas. The question arises whether or not they may represent separate populations, or whether they form subgroups which co-express more than one of these antigens. Therefore, the present study is focused on the distribution patterns of the axons single-immunolabelled by the catecholaminergic marker tyrosine hydroxylase (TH), and on TH-immunoreactive axons displaying also CR- and/or CALB-immunoreactivity in double-immunostained sections. Striking differences were found between the patch and matrix compartments of the caudate–putamen (CP). Whereas the vast majority of TH-immunoreactive fibres in the patches and a patch-associated subcallosal layer co-expressed CR but not CALB, fibres mono-labelled by the TH-immunoreactivity were predominant in the matrix. The matrix-like regions of the core of nucleus accumbens (CACC), fundus striati (FS), the striatal cell bridges (CB) and the striatal part of olfactory tubercle (OTU) coincided in this respect with the matrix in CP. The absence of CR-immunoreactivity was also characteristic of the TH-immunoreactive fibres in the patch-like areas of the accumbal core, although a high number of separate CR-immunoreactive axons were present. In the shell of nucleus accumbens (SACC) which receives a rich catecholaminergic innervation, fibres co-expressing either one of the calcium-binding proteins were absent. The islands of Calleja (CJI) displaying a strongly TH-immunoreactive centre and a periphery of lower staining intensity, showed only a low number of TH-immunoreactive fibres co-expressing CR or CALB. The broad shell-like band of TH-immunoreactive axons between medial and lateral part of the septum was single-stained with the TH-immunoreactivity. In contrast, the TH-positive fibres forming basket-like arrangements around some neurons in the dorsal lateral septal nucleus co-expressed also CR, but not CALB. The results are discussed in view of the recent concepts of basal forebrain organization and the cytochemical characteristics of mesencephalic dopaminergic nuclei giving rise to the vast majority of the striatal and septal TH-immunoreactive fibre supply, in order to correlate the known projection patterns with the content of calcium-binding proteins in TH-immunolabelled fibres and presumed cells of origin. The TH-immunoreactive fibres in the striatal patches displaying CR- but not CALB-immunoreactivity may originate mainly from neurons in the ventral tier of pars compacta (SNC) and from the pars reticulata of substantia nigra (SNR) which show identical cytochemical properties. Axons in the matrix of CP and the accumbal core as well as in the islands of Calleja single-labelled by the TH-immunoreactivity or additionally containing CALB and CR may originate from neurons in the dorsal tier of mesencephalic nuclei like SN, pars compacta and ventral tegmental area. CR-containing TH-immunoreactive basket-like axon terminations in the dorsal lateral septal nucleus are likely to originate either from mesencephalic nuclei or from the supramammillary region.

In vivo modulation of annexins I, II and V expression by thyroxine and methylthiouracil.

Regulation of annexin concentration and localization were investigated in thyroid tissues of hypothyroid [methylthiouracil (MeSur) treatment], euthyroid (control) and hyperthyroid [thyroxine (T4) treatment] rats. A low level of circulating thyroid hormones induces a decrease of total thyroid calcium-binding protein concentration when compared with the concentration in unstimulated animals. Conversely, concentrations of annexins I, II and V increase. The accumulation of these proteins in two subcellular compartments (cytosolic and particulate fractions) can be reversed by addition of thyroid hormones. The finding of a specific increase in annexins concentration in thyroid-hormone-deficient rats, with a general decrease of the total calcium-binding protein content points to a very important role of these proteins in the cells. Furthermore, hyperthyroidisnt gives opposite results. To investigate the transduction pathway of annexins I-, II- and V-induced biosynthesis by thyroid hormones in thyroid glands, we used cultured pig thyroid cells as in vitro model system. In previous work [16], we have shown that annexin concentrations and localization are under TSH control via the adenylate cyclase pathway. In the presence of MeSur (in the culture medium), the protein-binding iodine remains low, indicative of weak thyroid hormone synthesis (data not shown) and that the annexins content is unchanged. These results suggest that, in thyroid tissue, an indirect mechanism links thyroid hormones to annexin expressions via the TSH feed-back loop, and excludes autocrine regulation.

Hippocampal CA1 interneurons: an in vivo intracellular labeling study

Fast spiking interneurons in the CA1 area of the dorsal hippocampus were recorded from and filled with biocytin in anesthetized rats. The full extent of their dendrites and axonal arborizations as well as their calcium binding protein content were examined. Based on the spatial extent of axon collaterals, local circuit cells (basket and O-LM neurons) and long-range cells (bistratified, trilaminar, and backprojection neurons) could be distinguished. Basket cells were immunoreactive for parvalbumin and their axon collaterals were confined to the pyramidal layer. A single basket cell contacted more than 1500 pyramidal neurons and 60 other parvalbumin-positive interneurons. Commissural stimulation directly discharged basket cells, followed by an early and late IPSPs, indicating interneuronal inhibition of basket cells. The dendrites of another local circuit neuron (O-LM) were confined to stratum oriens and it had a small but high-density axonal terminal field in stratum lacunosum-moleculare. The fastest firing cell of all interneurons was a calbindin-immunoreactive bistratified neuron with axonal targets in stratum oriens and radiatum. Two neurons with their cell bodies in the alveus innervated the CA3 region (backprojection cells), in addition to rich axon collaterals in the CA1 region. The trilaminar interneuron had axon collaterals in strata radiatum, oriens and pyramidale with its dendrites confined to stratum oriens. Commissural stimulation evoked an early EPSP-IPSP-late depolarizing potential sequence in this cell. All interneurons formed symmetric synapses with their targets at the electron microscopic level. These findings indicate that interneurons with distinct axonal targets have differential functions in shaping the physiological patterns of the CA1 network.

Differential vulnerability of neurochemically identified subpopulations of retinal neurons in a monkey model of glaucoma

The vulnerability of subpopulations of retinal neurons delineated by their content of cytoskeletal or calcium-binding proteins was evaluated in the retinas of cynomolgus monkeys in which glaucoma was produced with an argon laser. We quantitatively compared the number of neurons containing either neurofilament (NF) protein, parvalbumin, calbindin or calretinin immunoreactivity in central and peripheral portions of the nasal and temporal quadrants of the retina from glaucomatous and fellow non-glaucomatous eyes. There was no significant difference between the proportion of amacrine, horizontal and bipolar cells labeled with antibodies to the calcium-binding proteins comparing the two eyes. NF triplet immunoreactivity was present in a subpopulation of retinal ganglion cells, many of which, but not all, likely correspond to large ganglion cells that subserve the magnocellular visual pathway. Loss of NF protein-containing retinal ganglion cells was widespread throughout the central (59–77% loss) and peripheral (96–97%) nasal and temporal quadrants and was associated with the loss of NF-immunoreactive optic nerve fibers in the glaucomatous eyes. Comparison of counts of NF-immunoreactive neurons with total cell loss evaluated by Nissl staining indicated that NF protein-immunoreactive cells represent a large proportion of the cells that degenerate in the glaucomatous eyes, particularly in the peripheral regions of the retina. Such data may be useful in determining the cellular basis for sensitivity to this pathologic process and may also be helpful in the design of diagnostic tests that may be sensitive to the loss of the subset of NF-immunoreactive ganglion cells.

Research Note: Responses of Laying Hens on Saline Drinking Water to Dietary Supplementation with Various Zinc Compounds

Production variables, eggshell defects, eggshell quality, the concentration of calcium-binding protein (CaBP), and the activity of carbonic anhydrase (CA) in the shell gland mucosa were determined in hens receiving town water (10 mg Na/L), or town water supplemented with 2 g NaCl/L (796 mg Na/L). Five treatments were examined. Control hens received town water and a proprietary layer mash containing .17% Na. The remaining four treatments received the water containing NaCl (2 g/L). Hens of Treatment 2 were fed the proprietary layer mash and those of Treatments 3, 4, and 5 received, respectively, the layer mash containing supplements of Zn-methionine (Zinpro-200; .5 g/kg), Zn sulfate (ZnSO4·7H2 O; .46 g/kg), or chelated Zn-EDTA (.54 g/kg) to supply the same concentration of Zn (.1 g/kg). The treatments were applied for 6 wk.Hens receiving the saline drinking water without any dietary Zn supplement produced significantly (P < .05) more eggs with shell defects than hens receiving the town water. This increase in the incidence of eggshell defects was associated with significant reductions in eggshell breaking strength, the concentration of CaBP, and the activity of CA. Supplementing the saline drinking water with any of the three Zn compounds significantly reduced the incidence of eggshell defects and in some cases improved shell breaking strength, the concentration of CaBP, and the activity of CA.

Parvalbumin and calbindin D-28K immunoreactivity in central ganglioglioma and dysplastic gangliocytoma of the cerebellum

Calbindin D-28K and parvalbumin immunocytochemistry were used in the study of central ganglionic cell tumors. Most neurons in the ganglioglioma were immunoreactive to calbindin D-28K, but a few cells were labeled with antibodies against parvalbumin. In contrast, most cells in dysplastic gangliocytoma of the cerebellum were parvalbumin immunoreactive, but fewer reacted with anti-calbindin antibodies. These latter cells had two or three dendrites with claw-shaped terminals and axons with recurrent collateral branches and varicose terminals filled with strings and buttons. These observations suggest that central ganglionic cell tumors, including dysplastic gangliocytoma of the cerebellum, are composed of neurons which, on the basis of their calcium-binding protein content, have particular metabolic and electrophysiological properties.

Calcium and Carbonate Supply in the Shell Gland of Hens Laying Eggs with Strong and Weak Shells and During and After a Rest from Lay

The concentration of calcium-binding protein (CaBP) and the activities of calcium adenosine triphosphatase (Ca2+-ATPase) and carbonic anhydrase (CA) were determined in the shell gland mucosa of hens in two experiments. In Experiment 1, laying hens on a proprietary layer mash were compared with hens rested from lay by the feeding of whole grain barley. In Experiment 2 comparisons were made of laying hens fed the proprietary layer mash and producing eggs with either strong or weak shells. These latter comparisons were also made when the shell gland was quiescent or active with respect to daily eggshell formation.Feeding whole grain barley reduced egg production to zero after 11 days. This reduction in rate of lay was accompanied by significant reductions in all three markers, the effect on Ca2+-ATPase and CaBP being less than for CA. Control values were regained between 10 and 16 days after the barley was replaced with the layer mash. Relative shell strength and the physiological status of the shell gland with respect to time of daily eggshell formation had no significant effect on any marker in Experiment 2.

Rat osteoblasts contain receptors for mineralocorticoid hormones

A protein, that binds calcium and is induced by vitamin D3, has been identified in chick duodenal mucosa. This calcium-binding protein (CaBP) has been separated from other proteins in the mucosal supernatant fraction and partially purified (30-fold), using dextran gel filtration (Sephadex G-100). Also, CaBP was shown to occur as a distinct electrophoretic band on acrylamide gel. CaBP was detected in all segments of the small intestine and kidney of the vitamin D3-treated rachitic chick but was not detectable by present methods in colon, liver, and muscle. Thus, the soft tissue localization of CaBP corresponds to known physiological sites of vitamin D3 action. In the small intestine, the concentration of CaBP in the vitamin D3-repleted chicks decreases in the order: duodenum > jejunum > ileum, which correlates with the relative abilities of these particular segments to absorb Ca. It is suggested that CaBP may be intimately involved in the translocation of Ca across the intestinal epithelium.

Gs-unit activity in vitamin D deficiency rickets

A protein, that binds calcium and is induced by vitamin D3, has been identified in chick duodenal mucosa. This calcium-binding protein (CaBP) has been separated from other proteins in the mucosal supernatant fraction and partially purified (30-fold), using dextran gel filtration (Sephadex G-100). Also, CaBP was shown to occur as a distinct electrophoretic band on acrylamide gel. CaBP was detected in all segments of the small intestine and kidney of the vitamin D3-treated rachitic chick but was not detectable by present methods in colon, liver, and muscle. Thus, the soft tissue localization of CaBP corresponds to known physiological sites of vitamin D3 action. In the small intestine, the concentration of CaBP in the vitamin D3-repleted chicks decreases in the order: duodenum > jejunum > ileum, which correlates with the relative abilities of these particular segments to absorb Ca. It is suggested that CaBP may be intimately involved in the translocation of Ca across the intestinal epithelium.

Pattern of neuronal death in the rat hippocampus after status epilepticus. Relationship to calcium binding protein content and ischemic vulnerability

The pattern of hippocampal cell death has been studied following hippocampal seizure activity and status epilepticus induced by 110-min stimulation of the perforant pathway in awake rats. The order of vulnerability of principal cells in the different hippocampal subfields—as determined by silver impregnation—was found to be very similar to the pattern found in ischemia; i.e., dentate hilus > CA1, subiculum > CA3c > CA3a,b > dentate granule cells. The hilar somatostatin-containing cells were the most vulnerable cell type, whereas all other subpopulations of nonprincipal neurons—visualized by immunocytochemistry for the calcium binding proteins parvalbumin and calbindin—were remarkably resistant. Pyramidal cells in the CA3 region containing neither of the examined calcium binding proteins were more resistant to overexcitation than CA1 pyramidal cells, most of which do contain calbindin. This indicates that no simple relationship exists between vulnerability in status epilepticus and neuronal calcium binding protein content, and that local and/or systemic hypoxia during status epilepticus may be responsible for the ischemie pattern of cell death.

Different populations of parvalbumin‐ and calbindin‐D28k‐immunoreactive neurons contain GABA and accumulate 3H‐D‐aspartate in the dorsal horn of the rat spinal cord

The colocalization of parvalbumin (PV), calbindin-D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H-D-aspartate selectively were investigated in neurons of laminae I-IV of the dorsal horn of the rat spinal cord. Following injection of 3H-D-aspartate into the basal dorsal horn (laminae IV-VI), perikarya selectively accumulating 3H-D-aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H-D-aspartate were found exclusively in laminae I-III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H-D-aspartate-labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H-D-aspartate. A high proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP-immunoreactive perikarya were GABA-negative. GABA-immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I-IV. A significant proportion of the GABA-negative but PV-immunoreactive neurons also showed CaBP-immunoreactivity in laminae II and IV. These results show that out of the two calcium-binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium-binding proteins in the dorsal horn of the rat spinal cord.

Non-pyramidal cells in the CA3 region of the rat hippocampus: relationships of fine structure, synaptic input and chemical characteristics

The non-pyramidal cells of the hippocampus are heterogeneous with respect to their morphology, peptide content, physiological properties, and postsynaptic targets. Here we demonstrate that the content of peptides (cholecystokinin, somatostatin) and calcium-binding proteins (parvalbumin and calbindin) of non-pyramidal cells is not related to a characteristic fine structure or synaptic input. Varying numbers of GABA-negative and GABA-positive input synapses of non-pyramidal cells indicate that these neurons are differently integrated in inhibitory and disinhibitory circuits.

Relationship of neuronal vulnerability and calcium binding protein immunoreactivity in ischemia

The relationship between neuronal calcium binding protein content (calbindin D28K: CaBP and parvalbumin: PV) and vulnerability to ischemia was studied in different regions of the rat brain using the four vessel occlusion model of complete forebrain ischemia. The areas studied, i.e. the hippocampal formation, neocortex, neostriatum and reticular thalamic nucleus (RTN), show a characteristic pattern of CaBP and PV distribution, and are involved in ischemic damage to different degrees. In the hippocampal formation CaBP is present in dentate granule cells and in a subpopulation of the CA1 pyramidal cells, the latter being the most and the former the least vulnerable to ischemia. Non-pyramidal cells containing CaBP in these regions survive ischemia, whereas PV-containing non-pyramidal cells in the CA1 region are occasionally lost. Hilar somatostatin-containing cells and CA3 pyramidal cells contain neither PV nor CaBP. Nevertheless, the latter are resistant to ischemia and the former is the first population of cells that undergoes degeneration. Supragranular pyramidal neurons containing CaBP are the most vulnerable cell group in the sensory neocortex. In the RTN the degenerating neurons contain both PV and CaBP. In the neostriatum, ischemic damage involves both CaBP-positive and negative medium spiny neurons, although the degeneration always starts in the dorsolateral neostriatum containing relatively few CaBP-positive cells. The giant cholinergic interneurons of the striatum contain neither CaBP nor PV, and they are the most resistant cell type in this area. These examples suggest the lack of a consistent and systematic relationship between neuronal CaBP or PV content and ischemic vulnerability. It appears that some populations of cells containing CaBP or PV are more predisposed to ischemic cell death than neurons lacking these proteins. These neurons may express high levels of calcium binding proteins because their normal activity may involve a high rate of calcium uptake and/or intraneuronal release.

Effects of altered thyroid states and undernutrition on the calbindin-D 28K (calcium-binding protein) content of the hippocampal formation in the developing rat

A quantitative study of calbindin-D 28K (calcium-binding protein) was carried out on the developing hippocampal formation in normal, hypothyroid, hyperthyroid, and underfed rats. In normal animals, the calbindin-D 28K content increased after birth in agreement with the distribution of the protein previously reported by immunocytochemistry. Calbindin-D 28K was strikingly spared, compared to the other proteins, from the effects of hypothyroidism. On the contrary, the calbindin-D 28K:protein ratio was transiently reduced by hyperthyroidism. Corrective doses of thyroxine to hypothyroid rats increased the calbindin-D 28K content whatever the period of the hormonal treatment, but they also had a marked effect on the hippocampal weight and the protein content, especially when the hormone was given on days 2–3. With this latter replacement therapy schedule, the calbindin-D 28K:protein ratio dropped from the high value of the hypothyroid animal to normal. Taken together, the results obtained in hypothyroidism, hyperthyroidism and replacement therapy are consistent with a pronounced action of thyroid hormone on hippocampal structures other than those containing calbindin-D 28K. Undernutrition, which, like hypo- or hyperthyroidism, also markedly impairs hippocampal growth, affected the calbindin-D 28K content per hippocampus but not the calbindin-D 28K:protein ratio. This emphasizes the unique influence of thyroid hormone on brain development. The relative preservation of calbindin-D 28K in the hippocampal formation of animals lacking thyroid hormone suggests that calbindin-D 28K function may be crucial in this brain region.

Calcium-binding protein in human duodenal biopsies.

Intestinal calcium-binding protein (CaBP) (molecular weight 10,000) was measured by a specific enzyme-linked immunoadsorbent assay in duodenal biopsies of 94 patients (aged 20-89 years). The patients were examined for complaints of upper abdominal dyspepsia, but no significant pathology was found by gastroduodenoscopy. The median amount of CaBP in duodenal biopsies was 6.0 micrograms/mg of cytosolic protein with a coefficient of variation of 0.6. No change in the amount of CaBP per mg of cytosolic protein was observed with age. A significant correlation (P less than 0.001) was found between the concentration of CaBP and s-1,25-dihydroxyvitamin D (1,25(OH)2D). The amount of CaBP per mg of cytosolic protein did not correlate with immunoreactive parathyroid hormone in serum, and no relation between CaBP and the specific activity of alkaline phosphatase of the mucosal biopsies was found. The results of the present study show a wide variation in the amount of the 10 kDa CaBP in duodenal biopsies of humans and no change with age. Further, a correlation between s-1,25(OH)2D and CaBP was found.

Physicochemical properties of calcium-binding protein isolated from rat liver cytosol: Ca2+-induced conformational changes.

The physicochemical properties of a calcium-binding protein (CaBP) isolated from rat liver cytosol was investigated. Isoelectric focusing in a polyacrylamide gel plate using the Broad pI Calibration Kit showed that the isoelectric point is 5.20. The ultraviolet (UV) absorption spectrum of CaBP showed a maximum at 278 nm. The conformational changes induced by Ca -binding to CaBP were examined by measuring the UV difference, fluorescence emission, and circular dichroism (CD) spectra. These spectra were altered by titration of CaBP with 1.0 mM Ca . The alterations could be attributed to an increased exposure of tyrosine and tryptophan residues to a more aqueous environment, resulting in an increased hydrophobicity of CaBP. From the CD spectrum, the apparent α-helical content of CaBP in Ca -free buffer was estimated to be 34%. This value was decreased by 1.0 mM Ca addition. The results suggest that Ca -binding induces conformational changes of CaBP, and that the protein contains distinct and specific ligand-binding sites for Ca .