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.

The Demonstration of Human Prostatic Acid Phosphatase (Pap) With Phosphorylcholine

1976 ◽  
Vol 34 ◽  
pp. 88-89
Author(s):  
José A. Serrano ◽  
Hannah L. Wasserkrug ◽  
Anna A. Serrano ◽  
Arnold M. Seligman
Keyword(s):  
Acid Phosphatase ◽  
Prostate Gland ◽  
Prostatic Acid Phosphatase ◽  
Human Prostate ◽  
Rat Tissues ◽  
Specific Substrate ◽  
Acid Phosphatases ◽  
Lysosomal Acid ◽  
Cacodylate Buffer

As previously reported (1, 2) phosphorylcholine (PC) is a specific substrate for prostatatic acid phosphatase (PAP) as opposed to other acid phosphatases, e.g., lysosomal acid phosphatase. The specificity of PC for PAP is due to the pentavalent nitrogen in PC, a feature that renders PC resistant to hydrolysis by all other acid phosphatases. Detailed comparative cytochemical results in rat tissues are in press. This report deals with ultracytochemical results applying the method to normal and pathological human prostate gland.Fresh human prostate was obtained from 7 patients having transurethral resections or radical prostatectomies. The tissue was fixed in 3% glutaraldehyde- 0.1 M cacodylate buffer (pH 7.4) for 15 min, sectioned at 50 μm on a Sorvall TC-2 tissue sectioner, refixed for a total of 2 hr, and rinsed overnight in 0.1 M cacodylate buffer (pH 7.4)-7.5% sucrose.

four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2767-2777 ◽  
Author(s):  
J.L. Villano ◽  
F.N. Katz
Keyword(s):  
Optic Lobe ◽  
Regional Growth ◽  
Positional Information ◽  
In Vitro Translation ◽  
Translation System ◽  
Regional Pattern ◽  
Microsomal Membranes ◽  
Position Sensitive ◽  
Coordination Function

Genes capable of translating positional information into regulated growth lie at the heart of morphogenesis, yet few genes with this function have been identified. Mutants in the Drosophila four-jointed (fj) gene show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with this coordination function. Consistent with a position-sensitive role, we show that fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. When the cDNA is translated in an in vitro translation system in the presence of exogenous microsomal membranes, the intralumenal portion of some of the molecules is cleaved, yielding a secreted C-terminal fragment. We propose that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of the imaginal discs.

scholarly journals Assessment of Acid Phosphatase Production by In vitro Cultures of Atropa acuminata

2016 ◽  
Vol 26 (1) ◽  
pp. 15-23
Author(s):  
Saima Khan ◽  
Meenu Katoch ◽  
Sharada Mallubhotla ◽  
Suphla Gupta ◽  
Manju Sambyal ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Period ◽  
Callus Cultures ◽  
Shoot Cultures ◽  
Crude Enzyme Extract ◽  
Atropa Acuminata

The potential of various culture lines of Atropa acuminata were investigated for resourcing acid phosphatase (ACP) (3.1.3.2). Crude enzyme extract comprised of a mixture of four isoforms, distinguishable by polyacrylamide gel electrophoresis (PAGE) with molecular weight ranging from 39 to 215 kDa. In vitro regenerated proliferative shoots, callus and roots showed higher specific activity (2.49, 3.41, 2.91 U/mg protein, respectively) as compared to in vivo grown plants (0.71 U/mg protein). ACP activity in root cultures increased progressively up to 4.6 U/mg during the entire growth period (2 ? 24 weeks), whereas in case of shoot cultures, the specific activity escalated to 2.49 U/mg at 8 weeks, which then declined subsequently (1.95 U/mg). Similarly, callus cultures initially showed a higher phosphohydrolytic activity (3.41 U/mg protein) until 8 weeks by which period, it decreased with the passage of growth period. The present studies reveal an alternate system for resourcing of ACP from Atropa acuminata.Plant Tissue Cult. & Biotech. 26(1): 15-23, 2016 (June)

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

1985 ◽  
Vol 58 (6) ◽  
pp. 2091-2095 ◽  
Author(s):  
T. E. Weaver ◽  
J. A. Whitsett ◽  
W. M. Hull ◽  
G. Ross
Keyword(s):  
Gel Electrophoresis ◽  
Pulmonary Surfactant ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surfactant Protein ◽  
Lung Lavage ◽  
Complex Carbohydrate ◽  
Monospecific Antiserum ◽  
Microsomal Membranes

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000–28,000 daltons; glycoprotein A2, 32,000–34,000 daltons; and glycoprotein A3, 37,000–38,000 daltons; pH at isoelectric point (pI) 4.5–5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8–5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000–34,000 daltons, pI 4.8–5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000–28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.

scholarly journals Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity

1989 ◽  
Vol 262 (3) ◽  
pp. 931-938 ◽  
Author(s):  
S M Smith ◽  
S E Kane ◽  
S Gal ◽  
R W Mason ◽  
M M Gottesman
Keyword(s):  
Molecular Mass ◽  
Proteolytic Activity ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Enzymic Activity ◽  
A431 Cells ◽  
Molecular Masses ◽  
Procathepsin L ◽  
Endoglycosidase H

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

scholarly journals The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

1986 ◽  
Vol 6 (12) ◽  
pp. 4478-4485 ◽  
Author(s):  
A T Garber ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Protein ◽  
Polyacrylamide Gel ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Alpha Cells ◽  
Two Dimensional ◽  
Isoelectric Ph

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains

1981 ◽  
Author(s):  
G M Fuller ◽  
J M Nickerson
Keyword(s):  
Signal Peptide ◽  
Hepatoma Cell ◽  
Temporal Analysis ◽  
In Vitro Translation ◽  
Translation System ◽  
Hepatoma Cell Line ◽  
Amino Terminal ◽  
Cellular Processes ◽  
Microsomal Membranes

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.

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 High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase

2012 ◽  
Vol 18 (4) ◽  
pp. 481-489 ◽  
Author(s):  
Eric S. McCoy ◽  
Wendy A. Lea ◽  
Bryan T. Mott ◽  
David J. Maloney ◽  
Ajit Jadhav ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
High Throughput ◽  
Cyclic Nucleotide ◽  
Prostatic Acid Phosphatase ◽  
Live Cells ◽  
Nucleotide Analogs ◽  
Small Molecule Modulators ◽  
Pharmacologically Active ◽  
Kinetic Mode

The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well–based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.

Sign in / Sign up

Export Citation Format

Share Document