scholarly journals Tartrate-resistant acid phosphatase from human osteoclastomas is translated as a single polypeptide

1991 ◽  
Vol 277 (3) ◽  
pp. 631-634 ◽  
Author(s):  
A R Hayman ◽  
A J Dryden ◽  
T J Chambers ◽  
M J Warburton
Keyword(s):  
Acid Phosphatase ◽  
Peptide Bond ◽  
Northern Blotting ◽  
Tartrate Resistant Acid Phosphatase ◽  
Acid Phosphatases ◽  
Post Translational Modification ◽  
Subunit Protein ◽  
Single Polypeptide ◽  
Single Peptide

Tartrate-resistant acid phosphatases have been isolated from a number of sources. These enzymes consist of one subunit (Mr 30,000-40,000) or two dissimilar subunits (Mr 15,000-20,000). Previously we isolated the enzyme from human osteoclastomas, as a two-subunit protein. By Northern blotting and hybridization with radiolabelled oligonucleotides corresponding to the N-terminal sequences of the two subunits, we demonstrate here that the enzyme is transcribed as one mRNA which is translated in vitro to produce a single polypeptide of approx. Mr 33,000. Transcription as a single mRNA species is also the case in other tissues. These results suggest that the osteoclastoma enzyme undergoes post-translational modification in the form of cleavage of a single peptide bond to give a disulphide-bonded two-subunit protein.

In vitro translation of human prostatic acid phosphatase mRNA and processing of the translation products by microsomal membranes and endoglycosidase H

1987 ◽  
Vol 65 (10) ◽  
pp. 921-924 ◽  
Author(s):  
Gilles Paradis ◽  
Jean Y. Dubé ◽  
Pierre Chapdelaine ◽  
Roland R. Tremblay
Keyword(s):  
Acid Phosphatase ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostatic Acid Phosphatase ◽  
In Vitro Translation ◽  
Acid Phosphatases ◽  
Major Band ◽  
Microsomal Membranes ◽  
Endoglycosidase H

Poly(A)+ RNA was isolated from human prostatic tissue and translated in vitro in a rabbit reticulocyte lysate translation assay. Acid phosphatase labeled with [35S]methionine was immunoprecipitated with an antibody against seminal plasma acid phosphatase. Two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitate, followed by fluorography, revealed the presence of two spots (one major and one minor), both having a molecular mass of 43 kilodaltons (kDa) and an isoelectric point higher than mature acid phosphatase. Addition of canine pancreatic membranes to the translation assay resulted in the formation of four immunoprecipitable spots with molecular masses ranging from 43 to 49 kDa on one-dimensional gels. These spots probably represent acid phosphatases containing one to four core sugar groups, since after the addition of endoglycosidase H the molecular mass heterogeneity was abolished and we observed only one major band with a molecular mass (41 kDa) slightly lower than the ones of the primary translation product. These results suggest that human prostatic acid phosphatases are synthesized as two 43-kDa preproteins, which are further processed to 41-kDa proteins by removal of their signal peptide. Heterogeneity of the native protein arises mostly from glycosylation at four sites and not from differences in the amino acid sequence of the various forms.

scholarly journals 1,25-Dihydroxy-19-nor-vitamin D2, a Vitamin D Analog with Reduced Bone Resorbing Activity In Vitro

2000 ◽  
Vol 11 (10) ◽  
pp. 1857-1864
Author(s):  
L. SHANNON HOLLIDAY ◽  
STEPHEN L. GLUCK ◽  
EDUARDO SLATOPOLSKY ◽  
ALEX J. BROWN
Keyword(s):  
Vitamin D ◽  
Acid Phosphatase ◽  
Bone Resorption ◽  
Vitamin D Receptor ◽  
Intrinsic Activity ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mouse Bone Marrow ◽  
Bone Resorbing Activity

Abstract. 1,25-Dihydroxy-19-nor-vitamin D2 (19-norD2), a new analog of 1,25(OH)2D3, suppresses parathyroid hormone in renal failure patients and in uremic rats but has less calcemic activity than 1,25(OH)2D3. Although 19-norD2 has high affinity for the vitamin D receptor and similar pharmacokinetics to those of 1,25(OH)2D3, it has much less bone resorbing activity in vivo. The intrinsic activity of 19-norD2 on osteoclastogenesis and activation of bone resorption in mouse bone marrow cultures was examined to determine the mechanism involved. 19-norD2 and 1,25(OH)2D3 (10 nM) were equivalent in stimulating the formation and maintenance of large multinucleated, tartrate-resistant acid phosphatase-positive cells. However, the amount of bone resorbed by osteoclasts stimulated by 10 nM 19-norD2, as measured by pit-forming assays, was reduced 62% compared with 10 nM 1,25(OH)2D3-stimulated osteoclasts (P < 0.05). This difference could not be attributed to enhanced catabolism or to downregulated vitamin D receptor. The rate of degradation of 19-norD2 in cultures was approximately 20% greater than 1,25(OH)2D3, not enough to account for the different effects on bone resorption. The VDR levels were identical in cultures that were treated with 19-norD2 and 1,25(OH)2D3. In summary, 19-norD2 is less effective than 1,25(OH)2D3 in stimulating mouse marrow osteoclasts to resorb bone. The reason for this difference is not clear but seems to involve the late maturation and/or activation of osteoclasts as the number of pits produced by each tartrate-resistant acid phosphatase-positive cell is reduced under stimulation by 19-norD2 compared with 1,25(OH)2D3.

Near-infrared fluorescence probe for the determination of acid phosphatase and imaging of prostate cancer cells

The Analyst ◽  
2015 ◽  
Vol 140 (5) ◽  
pp. 1629-1636 ◽  
Author(s):  
Zihan Lin ◽  
Ziping Liu ◽  
Hao Zhang ◽  
Xingguang Su
Keyword(s):  
Prostate Cancer ◽  
Acid Phosphatase ◽  
Cancer Cells ◽  
Near Infrared ◽  
Fluorescence Probe ◽  
Acid Phosphatases ◽  
Human Prostate Cancer ◽  
Prostate Cancer Cells

We had successfully applied the near-infrared CuInS2 QDs-based fluorescence acid phosphatases probe to perform in vitro imaging of human prostate cancer cells.

scholarly journals Functional Dissociation of the Basolateral Transcytotic Compartment from the Apical Phago-lysosomal Compartment in Human Osteoclasts

2005 ◽  
Vol 53 (5) ◽  
pp. 665-670 ◽  
Author(s):  
James Meagher ◽  
René Zellweger ◽  
Luis Filgueira
Keyword(s):  
Staphylococcus Aureus ◽  
Dendritic Cells ◽  
Acid Phosphatase ◽  
In Vitro Study ◽  
Infectious Agent ◽  
Tartrate Resistant Acid Phosphatase ◽  
Lysosomal Compartment ◽  
Acidic Compartment ◽  
Human Osteoclasts

Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. In dendritic cells, TRAP and bacteria were colocalized. In osteoclasts, there was no colocalization of bacteria, TRAP, or the acidic compartment, indicating that there are three distinct vesicular compartments: the apical phago-lysosomal compartment, the basal secretory compartment, and the basolateral transcytotic compartment. Dissociation of the TRAP-containing transcytotic vesicles from the apical phago-lysosomal compartment may restrain osteoclasts from eliminating S. aureus.

scholarly journals Nitrate reductase and acid phosphatase activities as affected by inorganic phosphate in corn roots

2014 ◽  
Vol 52 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Marie Kummerova ◽  
Józef Buczek
Keyword(s):  
Acid Phosphatase ◽  
Nitrate Reductase ◽  
Nutrient Solution ◽  
Inorganic Phosphate ◽  
Acid Phosphatases ◽  
Weak Effect ◽  
Corn Roots ◽  
Nr Activity

The deficieny of inorganic phosphate in nutrient solution reduces by about 50 per cent NO<sub>3</sub>- absorption in corn seedlings, it decreases both <em>in vitro</em> and in vivo nitrate reductase (NR) activity, as well the potential and actual NR level and has a very weak effect on NR induction. Acid phosphatases activities increase in corn roots when the plants are grown in nutrient solution without phosphorus. We suggest that inorganic phosphate is required mainly for maintenance of NR activity rather, than for induction <em>in vivo</em> of nitrate reductase. It is not excluded that deficiency of inorganic phosphate in root tissue may be partly supplemented as the result of enhanced acid phosphatase activity.

Overlapping functions of lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (Acp5) revealed by doubly deficient mice

Development ◽  
2001 ◽  
Vol 128 (23) ◽  
pp. 4899-4910
Author(s):  
Anke Suter ◽  
Vincent Everts ◽  
Alan Boyde ◽  
Sheila J. Jones ◽  
Renate Lüllmann-Rauch ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Tartrate Resistant Acid Phosphatase ◽  
Arylsulfatase A ◽  
Acid Phosphatases ◽  
Lysosomal Storage ◽  
Lysosomal Acid ◽  
Biochemical Analyses ◽  
Filamentous Material ◽  
Gait Disturbances ◽  
Deficient Mice

To date, two lysosomal acid phosphatases are known to be expressed in cells of the monocyte/phagocyte lineage: the ubiquitously expressed lysosomal acid phosphatase (LAP) and the tartrate-resistant acid phosphatase-type 5 (Acp5). Deficiency of either acid phosphatase results in relatively mild phenotypes, suggesting that these enzymes may be capable of mutual complementation. This prompted us to generate LAP/Acp5 doubly deficient mice. LAP/Acp5 doubly deficient mice are viable and fertile but display marked alterations in soft and mineralised tissues. They are characterised by a progressive hepatosplenomegaly, gait disturbances and exaggerated foreshortening of long bones. Histologically, these animals are distinguished by an excessive lysosomal storage in macrophages of the liver, spleen, bone marrow, kidney and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of Acp5- and LAP/Acp5-deficient mice. In osteoclasts of phosphatase-deficient mice, vacuoles were frequently found which contained fine filamentous material. The vacuoles in Acp5- and LAP/Acp5 doubly-deficient osteoclasts also contained crystallite-like features, as well as osteopontin, suggesting that Acp5 is important for processing of this protein. This is further supported by biochemical analyses that demonstrate strongly reduced dephosphorylation of osteopontin incubated with LAP/Acp5-deficient bone extracts. Fibroblasts derived from LAP/Acp5 deficient embryos were still able to dephosphorylate mannose 6-phosphate residues of endocytosed arylsulfatase A. We conclude that for several substrates LAP and Acp5 can substitute for each other and that these acid phosphatases are essential for processing of non-collagenous proteins, including osteopontin, by osteoclasts.

Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3151-3162 ◽  
Author(s):  
A.R. Hayman ◽  
S.J. Jones ◽  
A. Boyde ◽  
D. Foster ◽  
W.H. Colledge ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Endochondral Ossification ◽  
Bone Matrix ◽  
Axial Skeleton ◽  
Mutant Mice ◽  
Intrinsic Defect ◽  
Tartrate Resistant Acid Phosphatase ◽  
Long Bones ◽  
Electron Imaging

Mature osteoclasts specifically express the purple, band 5 isozyme (Acp 5) of tartrate-resistant acid phosphatase, a binuclear metalloenzyme that can generate reactive oxygen species. The function of Acp 5 was investigated by targeted disruption of the gene in mice. Animals homozygous for the null Acp 5 allele had progressive foreshortening and deformity of the long bones and axial skeleton but apparently normal tooth eruption and skull plate development, indicating a role for Acp 5 in endochondral ossification. Histomorphometry and mineralization density analysis of backscattered electron imaging revealed widened and disorganized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover. Increased mineralization density was observed in the long bones of older animals which showed modelling deformities at their extremities: heterozygotes and homozygous Acp 5 mutant mice had tissue that was more mineralized and occupied a greater proportion of the bone in all regions. Thus the findings reflect a mild osteopetrosis due to an intrinsic defect of osteoclastic modelling activity that was confirmed in the resorption pit assay in vitro. We conclude that this bifunctional metalloprotein of the osteoclast is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.

scholarly journals Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro.

1977 ◽  
Vol 73 (2) ◽  
pp. 492-504 ◽  
Author(s):  
D Raybin ◽  
M Flavin
Keyword(s):  
Neuroblastoma Cells ◽  
Peptide Bond ◽  
Bovine Brain ◽  
Post Translational Modification ◽  
Alpha Chain ◽  
Before And After ◽  
Tubulin Tyrosine Ligase ◽  
Rat Tissue

A post-translational modification of tubulin with potential regulatory significance has been revealed by the discovery of an enzyme (tubulin-tyrosine ligase) in brain extracts which can add a tyrosine residue to the alpha chain, apparently through peptide bond linkage to a C-terminal glutamate. We have investigated whether this modification also occurs in vivo, and whether it alters the extent to which tubulin can assemble in vitro. Cytoplasmic tubulin purified from bovine brain by cycles of assembly was shown to be partially tyrosylated. Carboxypeptidase A digestion of isolated alpha chains liberated about 0.3 equivalent of tyrosine. Brief digestion of native tubulin increased the proportion of alpha chains which could be tyrosylated by ligase, from 25 to 45%. The tubulin assembled to the same extent before and after carboxypeptidase treatment. When tubulin was purified after introducing labeled tyrosine with ligase, the labeled species assembled in the same proportion as unlabeled. Thus tubulin can be incorporated into microbubules in vitro with or without C-terminal tyrosine. An apparent resolution of alpha chain into two components by hydroxylapatite chromatography was shown not to be due to the presence or absence of C-terminal tyrosine. Tubulin-tyrosine ligase was found in extracts of every rat tissue examined, but was not detected in sea urchin eggs before or after fertilization, in Tetrahymena cells or cilia, or in yeast. Cultured neuroblastoma cells fixed tyrosine into tubulin alpha chains under conditions where protein synthesis was inhibited; this in vivo fixation appeared to be into an insoluble moiety of tubulin. Incidental to these studies, a new assay utilizing an enamine substrate for carboxypeptidase was investigated.

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