Presence Of Calcium-binding Protein Research Articles

Calcium-binding protein isolated from rat liver cytosol reverses activation of pyruvate kinase by Ca2+.

The effect of a calcium-binding protein (CaBP) isolated from rat liver cytosol on enzyme activation by Ca2+ was investigated. Pyruvate kinase (adenosine triphosphate : pyruvate 2-Ο- phosphotransferase; EC 2.7.1.40) isolated from liver cytosol was activated by addition of Ca2+ in the range of 1.0-103 μm; the concentration giving a half-maximal effect was 10 μm Ca2+. The enzyme activation by 10 μm Ca2+ addition was reversed by the presence of CaBP (4-40 μg/ml). With increasing concentrations of Ca2+ (1, 10 and 100, um Ca2+), the pyruvate kinase activity increased progressively. The increase of the enzyme activity by Ca2+ was clearly prevented by the presence of CaBP (20 μ/ml). CaBP had no effect on pyruvate kinase activity in the absence of Ca2+. Meanwhile, calmodulin (2.5-10 μg/ml) also reversed the activation of pyruvate kinase by Ca2+ (1 and 10 μM). These results indicate that CaBP may regulate the activation of pyruvate kinase by Ca2+. In particular, a novel CaBP may play an important role in the regulation of the Ca2+ effect on pyruvate kinase.

Effect of calcium-binding protein on the activation of phosphorylase a in rat hepatic particulate glycogen by Ca2+.

The effect of a calcium-binding protein (CaBP) isolated from rat liver cytosol on pnos-phorylase α activity in the hepatic particulate glycogen was investigated. The particulate glycogen phosphorylase α activity was significantly increased by addition of Ca2+ in the range of 1.0-103 μM. This increase was not prevented by the presence of N- (6-aminohexyl) -5-chloro-1-napthalenesulfonamide (W-7 100 μM), an inhibitor of calmodulin. The elevation of phosphorylase α activity by 10 μM Ca2+ addition was significantly inhibited by the presence of CaBP at a concentration of greater than 3.0 μM. This inhibition was complete at 7.0 μM CaBP. CaBP itself had no effect on the enzyme activity. Of various metals (20 μM) used, addition of Zn2+ caused a significant decrease of the particulate glycogen phosphorylase α activity, while Mg2+ Mn2+ and Cd2+ had no effect on the enzyme activity. The presence of 3.5 μM CaBP did not block the inhibition of the enzyme activity by Zn2+ addition, indicating that CaBP uniquely reverses the Ca2+ effect. The present study suggests that CaBP regulates the effect of Ca2+ on phosphorylase α activity in the hepatic particulate glycogen of rats.

Immunocytochemical detection of vitamin D-dependent calcium-binding protein (CaBP-28K) in vestibular sensory hair cells and vestibular ganglion neurones of the cat

Vestibular sensory hair cells, afferent fibres and vestibular ganglion neurones of the cat are intensely labelled by a specific antibody to rat kidney vitamin D-dependent calcium-binding protein (CaBP-28K). Type I hair cells are more weakly CaBP immunoreactive than type II hair cells. Ganglion neurones also present a differential staining. The presence of calcium-binding protein in sensory hair cells could be of interest for the understanding of transductional mechanisms.

Immunocytochemical detection of 28 000-MW calcium-binding protein in horizontal cells of the rat retina.

Horizontal cells of rat retina were labeled intensely by a specific antibody to cerebellar calcium-binding protein. The amacrine cells stained very weakly. The presence of calcium-binding protein in horizontal cells could be of interest for the understanding of the feedback action of these cells on photoreceptors.

Characterization of the three calcium binding proteins in the human placenta (author's transl)

Calcium binding protein (CaBP) has been reported to be involved in absorbing calcium in the duodenum. There is some evidence to show that active transport of calcium ions occurs from mother to fetus in the human placenta. The presence of CaBP in the human placenta was examined according to Taylor and Wasserman's method, extracting the vitamin D-dependent CaBP in the duodenum, and the character of CaBP was then studied. 38,000 X g supernatant of the villous homogenate of the human placenta was heated at 60 degrees C for 5 mins to neutralize the calcium binding activity resulting from the blood. The fractions containing CaBP were separated and partially purified on Sephadex G-100 and DEAE cellulose columns. The CaBP fractions were further purified by preparative agar electrophoresis. The Chelex-100-resin method was used for the detection of calcium binding activity in the eluates and for calculation of Kd values and of calcium binding sites. The molecular weights were determined by electrophoresis of SDS-polyacrylamide gel. Three CaBPs were found in the human placenta. The molecular weight, Kd (micro M) and the binding activity (Ca mol/CaBP mol) of the three placental CaBPs were CaBP-Peak I: 82,000, 0.64, 0.52; Peak II: 12,000, 0.67, 1.10; Peak III: 8,500, 0.75, 2.08, respectively. The tyrosine residue of CaBP has been shown to act as an important binding site in that removal of calcium ions from CaBP generates a blue-shifted phenomenon in the ultraviolet difference spectrum between 270 and 300 nm. However no difference spectrum was observed with the placental CaBPs. This result was further confirmed by amino acid analysis which showed that none of the placental CaBPs contained tyrosine, tryptophan, half-cystine and proline, which are usually found in most proteins. The circular dichronism (CD) spectrum of CaBP-Peak I in the far ultraviolet range showed two negative bands at 222 and 207 nm (alpha-helix structure), and removal of calcium ions caused no difference spectrum. CD spectra of CaBP Peak II and III in the far ultraviolet range revealed random coil structures in the presence and absence of bound calcium ions. These findings indicate that the calcium binding mechanism of the placental CaBPs should be different from that of the others. In this study, three kinds of human placental CaBP newly isolated were characterized according to molecular weights, Kd values and binding activities and clarified as having an amino acid composition quite different from the CaBPs already reported.

The ontogenesis of calcium-binding protein in fetal rat kidney.

The presence of calcium-binding protein (CaBP) in the nucleus and cytoplasm of epithelial cells of renal tubules varies with the physiologic state. As part of a study to determine whether or not intranuclear CaBP precedes intracytoplasmic CaBP in the same cell, we used peroxidase-labeled antibody against human renal CaBP to localize CaBP in fetal rat kidney tubules. This paper reports examination of kidneys from rats on each day of gestation from the 10th to term (21 days) and on each of the first seven postnatal days. CaBP was first detected in fetal rat kidneys on the 19th gestational day. The histochemical staining reaction that revealed the CaBP was less intense than that produced in kidneys from adult animals, but its distribution was like that in adults, with some cells having no CaBP, others having it in the cytoplasm only, in the nucleus only, or in both. By the seventh postnatal day the staining reaction was similar to the adult patterns in all respects.

Calcium-binding protein in the duodenal mucosa of uremic patients and normal subjects

In the present study we measured the content and determined the localization of a calcium-binding protein (CaBP) in the intestinal biopsy specimen of ten patients with severe renal insufficiency and in eight healthy individuals. In each patient a biopsy specimen of the iliac crest was obtained for evaluation of the degree of osteodystrophy. The CaBP was isolated from human kidneys. Its identity with human intestinal CaBP was suggested by acrylamid gel electrophoresis. Specific antibodies against it were developed in rabbits. The content of CaBP in duodenal mucosa biopsy material was measured by quantitative radial immunodiffusion. Its mean value in the duodenum of patients with renal insufficiency was 3.65 +/- 1.14 mug; in that of normal persons, 10.80 +/- 3.20 mug/mg of protein in the supernatant. There was neither a correlation between the type and duration of renal insufficiency and the CaBP content of the specimens nor between the degree of renal osteodystrophy and CaBP content. In normal persons the specific immunohistological activity indicating the presence of CaBP was found uniformly along the brush border and basement membrane of duodenal epithelial and the goblet cells. In uremic patients the fluorescence was markedly reduced in the typical locations, especially in the brush border area.

Determination of calcium binding protein in duodenum of rachitic babies before and after treatment with 25-hydroxycholecalciferol

Antiserum directed against calcium binding protein (CaBP) isolated from human kidneys was used for the immunofluorescent localization of CaBP in human intestine. Frozen sections of intestine obtained by biopsy from normal persons were tested by the indirect fluorescent antibody technique. Specific fluorescence indicating the presence of CaBP was observed at both the basal and apical poles of the intestinal absorptive cells while the goblet cells appeared to fluoresce non-specifically. Treatment of rachitic children with 25-hydroxyholecalciferol generally restored the pattern of fluorescence seen in intestinal tissue from normal persons. Concentration of CaBP as determined by the LAUREL-technique increades after treatment.

Localization of calcium-binding protein in intestinal tissue by immunofluorescence in normal, vitamin-D-deficient and uraemic subjects.

Antiserum directed against calcium-binding protein isolated from human kidneys was used for the immunofluorescent localization of calcium-binding protein in human intestine and kidney. Frozen sections of intestine obtained by biopsy from normal persons were tested by the indirect fluorescent antibody technique. Specific fluorescence indicating the presence of calciumbinding protein was observed at both the basal and apical poles of the intestinal absorptive cells while the goblet cells appeared to fluoresce non-specifically. Treatment of rachitic children with 25-hydroxycholecalciferol generally restored the pattern of fluorescence seen in intestinal tissue from normal persons. Examinations of intestinal biopsies from uraemic patients yielded variable results.