Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum

1988 ◽  
Vol 91 (1) ◽  
pp. 61-70 ◽  
Author(s):  
D.R. Macer ◽  
G.L. Koch
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Ionophore ◽  
Binding Activity ◽  
Calcium Binding Proteins ◽  
Molecular Weights

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich ‘shells’ followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.

Endoplasmic Reticulum and Calcium-Binding Proteins inLiriodendron TulipiferaL. (Magnoliaceae) Pollen

1994 ◽  
Vol 128 (1) ◽  
pp. 295-295 ◽  
Author(s):  
L. Navazio ◽  
L. Sponga ◽  
F. Guzzo ◽  
B. Baldan ◽  
P. Mariani ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Calcium Binding Proteins

scholarly journals Expression of the calcium-binding proteins MRP8 and MRP14 in monocytes is regulated by a calcium-induced suppressor mechanism

1994 ◽  
Vol 301 (3) ◽  
pp. 655-660 ◽  
Author(s):  
J Roth ◽  
M Goebeler ◽  
V Wrocklage ◽  
C van den Bos ◽  
C Sorg
Keyword(s):  
Blot Analysis ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Calcium Ionophore ◽  
Calcium Binding Proteins ◽  
Biologically Active ◽  
Inflammatory Factors ◽  
Transcriptional Level ◽  
Down Regulation ◽  
Biochemical Modification

MRP8 and MRP14 are two calcium-binding proteins of the S-100 family the expression of which is restricted to distinct stages of monocytic differentiation. Heteromeric MRP8/MRP14 complexes have been shown to represent their biologically active forms. However, it is not as yet clear whether biochemical modification of complexes, or regulation on the transcriptional level, are responsible for the control of MRP8/MRP14 expression. Employing Western-blot analysis and metabolic labelling we have demonstrated that patterns and metabolism of MRP8/MRP14 complexes do not change during up- or down-regulation of MRP8/MRP14. By Northern-blot analysis it was shown that MRP8/MRP14 are regulated at the transcriptional level rather than by biochemical modification of the complexes. Elevation of intracellular calcium levels by A23187, as well as by thapsigargin, was found to lead to specific down-regulation of MRP8/MRP14 mRNA which is in contrast with data reported for inflammatory factors such as interleukin-1 or tumour necrosis factor alpha. Concomitant application of actinomycin D and calcium ionophore indicated that this suppressive effect is mediated by decreased synthesis rather than increased degradation of MRP8/MRP14 mRNA. Finally, we demonstrated that calcium-mediated down-regulation of MRP8-MRP14 can be antagonized by cycloheximide, suggesting that a calcium-induced repressor protein is responsible for suppression of MRP8-MRP14 at the transcriptional level. Our data indicate that the function of MRP8-MRP14 is restricted to events associated with early stages of myelomonocytic activation.

Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding

2001 ◽  
Vol 357 (1) ◽  
pp. 83-95 ◽  
Author(s):  
Barbara KRAMER ◽  
David M. FERRARI ◽  
Peter KLAPPA ◽  
Nicole PÖHLMANN ◽  
Hans-Dieter SÖLING
Keyword(s):  
Calcium Binding ◽  
Binding Proteins ◽  
Peptide Binding ◽  
Binding Activity ◽  
Calcium Binding Proteins ◽  
Carboxypeptidase Y ◽  
Functional Roles ◽  
Independent Process

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b′ domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as ‘scrambled’ ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a′ domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b′ domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.

scholarly journals Calcium-binding proteins in the vorticellid spasmoneme

1978 ◽  
Vol 77 (2) ◽  
pp. 358-370 ◽  
Author(s):  
LM Routledge
Keyword(s):  
Molecular Weight ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Striated Muscle ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dry Mass ◽  
Calcium Binding Proteins ◽  
Polyacrylamide Gel

The proteins of the contractile spasmoneme from Vorticella convallaria, Carcheslium polypinum, and Zoothamnium geniculatum have been extracted in the detergent, sodium dodecyl sulfate (SDS), as well as urea and guanidine hydrochloride (GuCl). After SDS extraction, the molecular weight distribution of the proteins was examined by means of SDS-polyacrylamide gel electrophoresis. Significant amounts of material corresponding to the contractile proteins actin and tubulin are not present. The contractile organelles in the three species examined contain a group of closely related proteins of molecular weight near 20,000, which constitute a major part (40-60%) of the dry mass. The 20,000 mol wt proteins in Zoothamnium bind calcium with high affinity (pK congruent to 6) and are termed "spasmins." By means of urea polyacrylamide gel electrophorsis, it is demonstrated that in Carchesium and Zoothamnium certain spasmin components bind calcium even in the presence of 6 M urea. The binding of calcium in 6 M urea suggests a functional relationship between the spasmins and the calcium-binding proteins of striated muscle which behave similarly. The calcium binding in urea also indicates that the spasmins within a single spasmoneme have different calcium affinities, and this difference in calcium-binding properties may be an important factor in the physiological function of the organelle.

scholarly journals Calcium-ion-binding activity in human small-intestinal mucosal cytosol Purification of two proteins and interrelationship of calcium-binding fractions

1981 ◽  
Vol 197 (1) ◽  
pp. 55-65 ◽  
Author(s):  
M Davie
Keyword(s):  
Molecular Weight ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Mammalian Species ◽  
Binding Activity ◽  
Calcium Binding Proteins ◽  
Calcium Binding Protein ◽  
Small Intestinal Mucosa ◽  
Small Intestinal

Ca2+-binding activity was investigated in human small-intestinal mucosal cytosol. Binding was detected in fractions with molecular weights of 28000 and about 900000, as determined by gel filtration. No binding was found at molecular weight 12000-13000 (the molecular weight of calcium-binding protein in lower mammalian species) until the cytosol had been subjected to a hollow-fibre-filtration step. The appearance of Ca2+-binding at molecular weight 12000-13000 was associated with a decline in the 28000-mol.wt. calcium-binding fraction. The 12000-13000-mol.wt. fraction contained two distinct calcium-binding proteins. One of these proteins had properties similar to those of pig calcium-binding protein. Antiserum to this protein reacted against the 28000-mol.wt calcium-binding fraction in cytosol from human small-intestinal mucosa and from human kidney. An immunoassay method for one of the calcium-binding proteins was established. In normal duodenal mucosa the concentration was 915 micrograms/g and in the ileum it was 443 micrograms/g of mucosa. A subject with hypercalcaemic sarcoidosis had 1200 micrograms/g of mucosa in the jejunum, and a subject with an undetectable concentration of plasma 25-hydroxycholecalciferol had concentrations of calcium-binding protein in the mucosa similar to those found in normal subjects.

scholarly journals SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane.

1991 ◽  
Vol 266 (29) ◽  
pp. 19599-19610 ◽  
Author(s):  
I. Wada ◽  
D. Rindress ◽  
P.H. Cameron ◽  
W.J. Ou ◽  
J.J. Doherty ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Calcium Binding Proteins ◽  
Endoplasmic Reticulum Membrane

How Important are S100A8/S100A9 Calcium Binding Proteins for the Activation of Phagocyte NADPH Oxidase, Nox2

2009 ◽  
Vol 8 (4) ◽  
pp. 282-289 ◽  
Author(s):  
Sylvie Berthier ◽  
Athan Baillet ◽  
Marie-Helene Paclet ◽  
Philippe Gaudin ◽  
Francoise Morel
Keyword(s):  
Nadph Oxidase ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Calcium Binding Proteins ◽  
Phagocyte Nadph Oxidase
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