scholarly journals Megakaryocyte maturation indicated by methanol inhibition of an acid phosphatase shared by magakaryocytes and platelets

Blood ◽  
1977 ◽  
Vol 50 (5) ◽  
pp. 905-914 ◽  
Author(s):  
OS Markovic ◽  
NR Shulman
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Fast Moving ◽  
Polyacrylamide Gel Electrophoresis ◽  
Optimal Conditions ◽  
Methanol Inhibition ◽  
Cytochemical Demonstration ◽  
Slow Moving ◽  
Moving Band ◽  
Platelet Formation

Abstract Optimal conditions necessary for the cytochemical demonstration of megakaryocyte (Mk) and platelet acid phosphatase (AP) were determined. Methanol, a constituent of fixatives commonly used in AP cytochemistry, was found to inhibit MkAP, and the degree of inhibition varied with Mk maturity. Immature Mk contained predominantly methanol-resistant AP, and mature Mk, predominantly methanol-sensitive AP. Platelets contained methanol-sensitive AP similar to mature Mk, suggesting that this enzyme provides an index of platelet formation by Mk. Soluble platelet AP showed three bands on polyacrylamide gel electrophoresis, visualized by the same reactions applied cytochemically. Two bands, accounting for 98% of the platelet AP activity, were slow moving and methanol sensitive; and one fast moving band accounting for 2% of activity was methanol resistant. Measurement of Mk and platelet AP isoenzymes may prove to have applications in evaluating Mk function.

scholarly journals Megakaryocyte maturation indicated by methanol inhibition of an acid phosphatase shared by magakaryocytes and platelets

Blood ◽  
1977 ◽  
Vol 50 (5) ◽  
pp. 905-914
Author(s):  
OS Markovic ◽  
NR Shulman
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Fast Moving ◽  
Polyacrylamide Gel Electrophoresis ◽  
Optimal Conditions ◽  
Methanol Inhibition ◽  
Cytochemical Demonstration ◽  
Slow Moving ◽  
Moving Band ◽  
Platelet Formation

Optimal conditions necessary for the cytochemical demonstration of megakaryocyte (Mk) and platelet acid phosphatase (AP) were determined. Methanol, a constituent of fixatives commonly used in AP cytochemistry, was found to inhibit MkAP, and the degree of inhibition varied with Mk maturity. Immature Mk contained predominantly methanol-resistant AP, and mature Mk, predominantly methanol-sensitive AP. Platelets contained methanol-sensitive AP similar to mature Mk, suggesting that this enzyme provides an index of platelet formation by Mk. Soluble platelet AP showed three bands on polyacrylamide gel electrophoresis, visualized by the same reactions applied cytochemically. Two bands, accounting for 98% of the platelet AP activity, were slow moving and methanol sensitive; and one fast moving band accounting for 2% of activity was methanol resistant. Measurement of Mk and platelet AP isoenzymes may prove to have applications in evaluating Mk function.

Isolation and Characterization of J-chain from Bovine Colostral Immunoglobulin M

1975 ◽  
Vol 53 (9) ◽  
pp. 943-949 ◽  
Author(s):  
R. Komar ◽  
T. K. S. Mukkur
Keyword(s):  
Gel Electrophoresis ◽  
Fast Moving ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Immunoglobulin M ◽  
J Chain ◽  
Isolation And Characterization ◽  
Moving Band ◽  
Covalently Bound

Purified bovine colostral intact immunoglobulin M (IgM) exhibited the presence of an anodal, single, fast moving band (noncovalently bound form) when subjected to analytical polyacrylamide gel electrophoresis at an alkaline pH in urea. Reduced and alkylated or sulfitolysed bovine colostral IgM (devoid of the noncovalently bound form) also showed the presence of a similar band (covalently bound form). The molecular weight of both the covalently bound and noncovalently bound forms of the fast component was determined to be 16 500 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. In addition, the non-covalently bound form of the fast-moving component was found to be antigenically identical to the covalently bound form. The noncovalently bound form sedimented as a single peak at 1.56 S. Antiserum against the fast-moving component precipitated neither bovine colostral IgG nor μ-chains and bovine serum albumin, but precipitated native or denatured intact IgM (devoid of the non-covalently bound form) and human J-chains and vice versa, thus permitting the fast-moving components to be classified as J-chains. Radioalkylation experiments revealed the presence of 9.7 sulfhydryl groups per mole, for both the covalently and non-covalently bound forms of bovine J-chain. The stoichiometry of J-chain, determined from the densitometric tracing of the reduced and alkylated bovine colostral IgM (devoid of the noncovalently bound J-chain) in stained analytical polyacrylamide gels, revealed the presence of one J-chain per IgM molecule. On the other hand the amount of non-covalently bound form of J-chain was determined to be 1.2 per molecule of IgM.

EFFECTS OF 5α-ANDROSTANE-3β,17β-DIOL AND 5β-DIHYDROTESTOSTERONE ON ACID PHOSPHATASE ACTIVITY IN THE PROSTATE GLAND OF THE CASTRATED ADULT RAT

1978 ◽  
Vol 79 (1) ◽  
pp. 9-16 ◽  
Author(s):  
M. P. TENNISWOOD ◽  
PAMELA P. ABRAHAMS ◽  
C. E. BIRD ◽  
A. F. CLARK
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Gel Electrophoresis ◽  
Acid Phosphatase Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostate Gland ◽  
Intraperitoneal Administration ◽  
Adult Rat ◽  
Secretory Acid Phosphatase

Polyacrylamide gel electrophoresis of filtrates from adult rat prostatic tissue showed two bands of acid phosphatase activity. These corresponded to the lysosomal and secretory acid phosphatases. After castration the secretory acid phosphatase disappeared. The specific activity of the enzyme increased from the time of castration to a maximum on day 7 before declining steadily, while the percentage inhibition by tartrate of acid phosphatase increased from control levels to a maximum on day 7 and then decreased to a new steady state by day 15. When 5α-androstane-3β,17β-diol was administered i.p. at a dose of 2 mg/day, starting immediately after castration, the secretory acid phosphatase was retained but the percentage inhibition and the specific activity were both raised above control levels. When this steroid was administered daily starting 7 days after castration the secretory acid phosphatase band on the gels returned more rapidly than with the classical androgens, but the percentage inhibition and specific activity were once again raised. Intraperitoneal administration of 5β-dihydrotestosterone, at a dose of 2 mg/day, did not maintain the secretory acid phosphatase activity which disappeared by day 5. However, the specific activity of acid phosphatase and the percentage inhibition by tartrate were both raised throughout the experiment. If this steroid was given 7 days after castration, the percentage inhibition by tartrate did not respond and fell to the level seen in castrated rats. The specific activity, however, remained significantly above the level found in castrated control rats.

Human Prostatic Acid Phosphatase: Properties of the Native Enzyme, and the Enzyme-Antibody Complex

1983 ◽  
Vol 20 (6) ◽  
pp. 374-380 ◽  
Author(s):  
Renze Bais ◽  
Anne Huxtable ◽  
John B Edwards
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostatic Acid Phosphatase ◽  
Native Enzyme ◽  
Terminal Amino Acid ◽  
Large Molecular Weight ◽  
Antibody Complex ◽  
Terminal Amino

Acid phosphatase purified from human prostatic tissue was shown to be homogeneous by polyacrylamide gel electrophoresis and N-terminal amino acid analysis. However, isoelectric focusing revealed a large number of isoenzymes which were reduced to four by digestion with neuraminidase. It is suggested that the patterns observed are due to differences in bound carbohydrate attached to the same protein backbone. Antiserum to the purified enzyme was produced in rabbits and reacted with the enzyme to form an enzymatically active complex of large molecular weight. This complex is more stable at high temperatures than the native enzyme. Kinetic analysis of both the enzyme and the enzyme-antibody complex demonstrated that the binding of the antibody caused no significant change to the active site of the enzyme.

scholarly journals New form of acid phosphatase during lysosome biogenesis

1981 ◽  
Vol 198 (1) ◽  
pp. 9-15 ◽  
Author(s):  
G R Rao ◽  
H N Aithal ◽  
F G Toback ◽  
G S Getz
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Lysosomal Enzyme ◽  
Gel Electrophoresis ◽  
Acid Phosphatase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Deficient Diet ◽  
Lysosomal Fraction ◽  
K Depletion

Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity.

scholarly journals Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas

1989 ◽  
Vol 261 (2) ◽  
pp. 601-609 ◽  
Author(s):  
A R Hayman ◽  
M J Warburton ◽  
J A S Pringle ◽  
B Coles ◽  
T J Chambers
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Tartrate Resistant Acid Phosphatase ◽  
Bovine Bone ◽  
Ph Optimum ◽  
Original Tumour

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.

scholarly journals Immunologically reactive tryptic fragments of human prostatic acid phosphatase

1984 ◽  
Vol 223 (3) ◽  
pp. 871-877 ◽  
Author(s):  
C L Lee ◽  
S S L Li ◽  
T M Chu
Keyword(s):  
Acid Phosphatase ◽  
Inhibitory Activity ◽  
Gel Electrophoresis ◽  
Active Sites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Prostatic Acid Phosphatase ◽  
Peptide Fragments

Three peptide fragments (designated II, III and IV) of human prostatic acid phosphatase (PAP) were isolated to homogeneity from a limited tryptic hydrolysate of PAP by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose and Sephadex G-75. The homogeneity was confirmed by disc poly-acrylamide-gel electrophoresis. The Mr values were 32 500, 25 000 and 11 000 as estimated by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation study revealed that only fragment II formed an immune precipitate with anti-PAP antibodies. Fragment II exhibited 45% of maximum inhibitory activity on the reaction between PAP and goat anti-PAP IgG (immunoglobulin G) antibodies (or rabbit anti-PAP antibodies), whereas fragments III and IV demonstrated 24% (or 23%) and 29% (or 27%) inhibition respectively. A mixture of these three tryptic fragments of PAP result in 96% (for goat anti-PAP antibodies) and 94% (for rabbit anti-PAP antibodies) inhibitory activities, which were equivalent to the sum of maximum inhibitory activity of the three fragments individually. The results demonstrated that these three tryptic peptide fragments carried all the antigenic active sites of the native PAP, and suggested that the entire molecule of human PAP comprised a minimum of four distinguishable, nonoverlapping antigenic determinants. These three fragments also were shown to retain all the disulphide bonds of the native PAP, and thus were useful reagents for the elucidation of PAP molecular structure.

scholarly journals Characterization of Filbert (Corylus) Species and Cultivars Using Gradient Polyacrylamide Gel Electrophoresis

1987 ◽  
Vol 5 (1) ◽  
pp. 11-16
Author(s):  
Z. Ahmad ◽  
L.S. Daley ◽  
R.A. Menendez ◽  
H.B. Lagerstedt
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phenol Oxidase ◽  
Chemical Identification ◽  
Identification Procedure ◽  
Genetic Characteristics ◽  
Isozyme Patterns ◽  
Anionic Polyacrylamide

A chemical identification procedure previously used to identify apple and pear species, cultivars and clonal accessions, was tried with Corylus (filbert, hazel) species, cultivars and clonal accessions. Following electrophoresis, the peroxidase, phenol oxidase, and acid phosphatase isozyme patterns on anionic polyacrylamide gradient gels were determined. These patterns were found to vary between clonal accessions, but did not change, within a given accession during and following the test period (May through October). Thus, these patterns were considered to represent genetic characteristics suitable for identification purposes. The patterns were used to identify 78 Corylus accessions at the National Clonal Germplasm Repository Corvallis, Oregon. All accessions tested (species, cultivars and clones) were distinguishable using this system. The diversity of isozyme patterns was greater in Corylus than Pyrus populations previously sampled. This technique appears to have the potential to readily identify filbert accessions and could be an important aid in the characterization of germplasm material.

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