Rat prostatic acid phosphatase: androgenic control of isoelectric focusing patterns

1983 ◽  
Vol 61 (7) ◽  
pp. 744-749 ◽  
Author(s):  
J. Downey ◽  
D. Mahan ◽  
T. G. Flynn ◽  
C. E. Bird ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Isoelectric Focusing ◽  
Specific Activity ◽  
Electrophoretic Pattern ◽  
Prostatic Acid Phosphatase ◽  
Staining Intensity ◽  
Adult Rats ◽  
Enzyme Specific Activity ◽  
Ph Range

To further characterize the androgen dependence of prostatic acid phosphatase (AP), the isoelectric focusing patterns of enzyme activity have been examined for normal and castrated adult rats and for rats receiving androgen injections. Isoelectric focusing was performed in polyacrylamide gels over the pH range 4–8. Naphthyl phosphate was used as substrate for staining. For normal rats there was a single lysosomal band (isoelectric point (pI) = 7.35 ± 0.04), four closely migrating secretory bands (pI = 5.96–5.63), and an androgen-dependent band (pI = 6.37 ± 0.05) which as yet has not been identified as either lysosomal or secretory. Following castration the secretory bands decreased significantly in staining intensity, the androgen-dependent band disappeared, and two new lysosomal bands (pI's = 7.13 ± 0.03 and 7.00 ± 0.03) appeared. With androgen replacement the latter two bands disappeared, the androgen-dependent band reappeared, and the secretory bands increased in staining intensity but with the most anodic of the four appearing before the others. This suggests that it could be a precursor to the others. The isoelectric focusing patterns of AP activity appear to be a better method of assessing the androgen status of the prostate than are the previously used parameters, namely, enzyme specific activity, degree of inhibition by tartrate, and polyacrylamide gel electrophoretic pattern.

Isolation and characterization of electrophoretic variants of human prostatic acid phosphatase.

1983 ◽  
Vol 29 (11) ◽  
pp. 1886-1889 ◽  
Author(s):  
M D Hibbard ◽  
R C McCarthy ◽  
H Markowitz
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Specific Activity ◽  
Prostatic Acid Phosphatase ◽  
Ph Gradient ◽  
Electrophoretic Variants ◽  
Isolation And Characterization ◽  
Immunochemical Determination ◽  
Km Value ◽  
Specific Activities

Abstract Prostatic acid phosphatase (EC 3.1.3.2) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.

Purification of human prostatic acid phosphatase by affinity chromatography and isoelectric focusing. Part I.

1978 ◽  
Vol 24 (3) ◽  
pp. 466-470 ◽  
Author(s):  
P Vihko ◽  
M Kontturi ◽  
L K Korhonen
Keyword(s):  
Acid Phosphatase ◽  
Sodium Dodecyl Sulfate ◽  
Affinity Chromatography ◽  
Isoelectric Focusing ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Prostatic Acid Phosphatase ◽  
Polyacrylamide Gel ◽  
Dodecyl Sulfate

Abstract The main isoenzyme of human prostatic acid phosphatases was purified by affinity chromatography on L(+)-tartrate linked to agarose and by isoelectric focusing. The enzyme was a single protein when examined by polyacrylamide gel electrophoreses, either as a native protein or in the presence of sodium dodecyl sulfate. The analytical recovery of enzyme activity was 19%. The specific activity was 40 18 mumol/(min X mg) for hydrolysis of 5.5 mmol/liter p-nitrophenyl phosphate at pH 4.8 and 37 degrees C. The purification factor was as great as 1900. The molecular weight of the enzyme as measured by gel filtration was 109 000 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate 54 000, indicating that the enzyme had been isolated in the dimer form. By this method we have achieved the best purification of human prostatic acid phosphatase so far.

Prostatic Acid Phosphatase: Comparison of Radioimmunoassay and Enzyme Activity Assay

1983 ◽  
Vol 129 (6) ◽  
pp. 1136-1140 ◽  
Author(s):  
H.J.A. Mensink ◽  
J. Marrink ◽  
F.R. Hindriks ◽  
A.K. van Zanten
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Prostatic Acid Phosphatase ◽  
Activity Assay ◽  
Enzyme Activity Assay

scholarly journals Studies on the development of ornithine–keto acid aminotransferase activity in rat liver

1968 ◽  
Vol 108 (4) ◽  
pp. 521-525 ◽  
Author(s):  
Niels C. R. Räihä ◽  
M. P. Kekomäki
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Single Injection ◽  
Repeated Administration ◽  
Natural Increase ◽  
Activity Levels ◽  
Keto Acid ◽  
Aminotransferase Activity ◽  
Adult Rats ◽  
Transient Elevation

1. During the normal development of the rat, the specific activity of liver ornithine–keto acid aminotransferase exhibits a transient elevation around term, and subsequently increases to adult activity levels during the third postnatal week. 2. The synthetic glucocorticoid triamcinolone, administered as a single injection, produces a marked elevation of the ornithine–keto acid aminotransferase activity within 24hr. if given postnatally before the natural increase in ornithine–keto acid aminotransferase activity has occurred. In foetal and adult animals, triamcinolone does not induce any increase in this enzyme activity. 3. The repeated administration of puromycin completely prevents the rise in ornithine–keto acid aminotransferase activity that follows triamcinolone administration. 4. If adult rats are fed with a protein-free carbohydrate diet, or one free of arginine, the ornithine–keto acid aminotransferase activity diminishes to a fraction of the normal. When such diets are given, a single injection of triamcinolone does not increase the enzyme activity within 24hr. 5. Partial hepatectomy, and repeated injections of growth hormone, depress the ornithine–keto acid aminotransferase activity in adult rats. 6. The findings are discussed in relation to the mechanisms concerned with developmental and adaptive changes in enzyme activities in the liver.

A quantitative study of the fixation of acid phosphatase by formaldehyde and its relevance to histochemistry

1974 ◽  
Vol 186 (1083) ◽  
pp. 137-164 ◽  
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Relative Activity ◽  
Buffer System ◽  
Inhibitory Effects ◽  
Fixed Tissue ◽  
Apparent Loss ◽  
Nitrophenyl Phosphate ◽  
Enzyme Specific Activity ◽  
Tissue Nitrogen

Fixatives reduce the activities of most enzymes in sections and blocks of tissue, but pre­vious studies do not agree by how much. In this paper it is shown that the disagreement is due principally to the use of inappropriate measurement parameters and to variations in the way tissues are prepared. Some new approaches to this problem are reported. They are illustrated by measurements on the activities of acid phosphatase (against p -nitrophenyl phosphate) remaining in blocks and cryostat-cut sections of hamster kidney after fixation in buffered solutions of formaldehyde. Whole fresh kidneys were cut into 1 mm 3 cubes. Two or more ‘lots’ of 9-12 cubes were chosen at random, fixed, washed, homogenized and assayed for residual enzyme specific activity which was expressed in terms of tissue nitrogen content or frozen-dried mass rather than protein content. The activity apparently remaining after fixation depends on, it was found, the time and temperature of fixation, the buffer salt in the fixative, the length of the post-fixation wash, and the duration and temperature of homogenization. For example, the relative activity (that is, specific activity expressed as a percentage of that of unfixed tissue) of acid phos­phatase in cubes assayed immediately after fixation is 46%; after a 24 h post-fixation wash in cold buffer it rises to 84%. Fixation causes only a 2% loss of nitrogenous material from the cubes. A further 2.7% is lost during the 24 h wash. Thus, much of the apparent loss of enzyme specific activity in fixed tissue is due to the inhibitory effects of fixative remaining in the tissue. Similar inhibition effects were observed in cryostat-cut sections, although sections frozen-dried on to coverslips showed a greater tolerance than fresh ones. For example, after 5 min fixation and without a post-fixation wash, the activity of acid phosphatase in fresh, cryostat-cut, mounted sections falls by about 30%, but that in frozen-dried ones is un­affected. After 25 min fixation, however the activity in the frozen-dried sections drops to about 84% of that of the unfixed tissue. A nitrogen-free buffer system, 5-norbornene-2, 3-dicarboxylic acid-NaOH, was used as the fixative vehicle for much of this study. A higher acid phosphatase activity is retained in tissues fixed or stored in this buffer than in either cacodylate or phosphate.

Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states

1979 ◽  
Vol 57 (5) ◽  
pp. 396-401 ◽  
Author(s):  
Hsiao-Lin Chang ◽  
Darold Holten ◽  
Rom Karin
Keyword(s):  
Enzyme Activity ◽  
Mammary Gland ◽  
Rat Liver ◽  
Specific Activity ◽  
Molecular Forms ◽  
Polyacrylamide Gels ◽  
Multiple Molecular Forms ◽  
Pregnancy And Lactation ◽  
Enzyme Specific Activity ◽  
Glucose 6 Phosphate Dehydrogenase

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland), it was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.

POSTNATAL CHANGES OF ACTIVITY AND ELECTROPHORETIC PATTERN OF JEJUNAL AND ILEAL NONSPECIFIC ESTERASE AND ALKALINE PHOSPHATASE OF THE RAT: EFFECT OF ADRENALECTOMY

1967 ◽  
Vol 45 (9) ◽  
pp. 1375-1384 ◽  
Author(s):  
H. Pelichová ◽  
O. Koldovský ◽  
A. Heringová ◽  
V. Jirsová ◽  
J. Kraml
Keyword(s):  
Alkaline Phosphatase ◽  
Postnatal Development ◽  
Esterase Activity ◽  
Specific Activity ◽  
Electrophoretic Pattern ◽  
Nonspecific Esterase ◽  
Enzyme Specific Activity ◽  
E 600 ◽  
Nonspecific Esterase Activity ◽  
Adrenalectomized Rats

During postnatal development of the rat, the activity of nonspecific esterase (substrate, β-naphthyl acetate) in the jejunum and ileum increased. Electrophoresis showed that distribution of this enzyme was different in these two parts of the intestine in the suckling rat, whereas the zymogram was the same for both parts in the adult rat. In intact animals, the degree of inhibition of nonspecific esterase activity by 10−4 M eserin, 5 × 10−8 M di-ethyl p-nitrophenyl phosphate (E.600), and 0.4 M NaCl was different, but there were no differences with any one inhibitor between the jejunum and the ileum of suckling and weanling rats. Adrenalectomy at 7 days of age did not influence the activity of the enzyme, but at 14 days of age it inhibited the rise of activity in jejunum and ileum. Adrenalectomy influenced the sensitivity of nonspecific esterase activity to inhibition by eserin and E.600. When cortisone was administered to adrenalectomized rats, certain characteristics of the enzyme (specific activity, sensitivity to various inhibitors, zymogram) were, in most case, comparable with those of preparations from normal controls.During postnatal development, the activity of alkaline phosphatase (substrate, β-naphthyl phosphate) in the jejunum showed practically no change, but in the ileum it decreased. Change in the zymogram was of the same type in both jejunum and ileum. Adrenalectomy at 7 days of age was followed by a fall in activity in the ileum that was apparent at 14 days of age, but the zymogram was not affected. Adrenalectomy on day 14 did not produce an apparent change by day 21, but there was retention of the earlier electrophoretic pattern. When cortisone was administered to adrenalectomized rats, certain characteristics of the enzyme (specific activity, zymogram) were comparable with those of preparations from normal controls.The results are discussed from the point of view of difference between the jejunum and ileum of suckling animals, and the influence of the adrenal glands.

scholarly journals Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme

1998 ◽  
Vol 331 (3) ◽  
pp. 929-935 ◽  
Author(s):  
Anette HENKER ◽  
Ilka SCHINDLER ◽  
Andreas RENZ ◽  
Erwin BECK
Keyword(s):  
Isoelectric Focusing ◽  
Spinacia Oleracea ◽  
Specific Activity ◽  
Dissociation Constants ◽  
Protein Band ◽  
Enzyme Substrate ◽  
Spinacia Oleracea L ◽  
Starch Debranching Enzyme ◽  
Ph Range ◽  
The Individual

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1.41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1–7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands (‘substrate-induced forms’, numbered I–III) with different specific activities, dissociation constants of the enzyme–substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.

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