Showing papers on "Alkaline phosphatase published in 1969"

Ectopic Production of an Alkaline Phosphatase Isoenzyme in Patients with Cancer

Abstract: An alkaline phosphatase isoenzyme (Regan isoenzyme) has been identified in the serum of 27 patients with various malignant tumors. This isoenzyme is biochemically and immunologically indistinguishable from placental alkaline phosphatase. In addition to being present in serum, it has been identified in both tumor tissue and malignant effusion fluids. Measurement of this isoenzyme has been clinically useful in monitoring progression or regression of tumor, identifying a source of serum alkaline phosphatase elevation, and identifying malignant effusions.

PLASMA MEMBRANES OF THE RAT LIVER: Isolation and Enzymatic Characterization of a Fraction Rich in Bile Canaliculi

Abstract: A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5'-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg++, 5'-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na+-K+-activation of ATPase is observed to the extent of 20–36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.

Degranulation of leukocytes in chronic granulomatous disease

Abstract: Quantitative chemical analyses of the subcellular distribution patterns for acid and alkaline phosphatase, beta glucuronidase and peroxidase were obtained for human peripheral blood leukocytes of four patients with chronic granulomatous disease (CGD). Five young adults with acute infections served as controls. The observations were made on fractions obtained by homogenization and centrifugation of leukocytes previously incubated with or without particles for ingestion. Distributions in resting CGD and normal cells were very similar for acid and alkaline phosphatase and peroxidase, but the proportion of beta glucuronidase in the granule fraction of CGD cells was depressed, with an increased proportion in the soluble fraction. Release of granule-bound enzymes during phagocytosis of a variety of particles was the same for CGD and control cells, except that release of beta glucuronidase was less marked in CGD cells. Total enzymatic activity of CGD cells for the hydrolases studied was normal. The data indicated that granular enzymes are released in a normal fashion in phagocytizing CGD cells. Supportive evidence of release of enzymes into the phagocytic vacuole of CGD cells was obtained by an electron microscopic study of myeloperoxidase.

Distribution of acid and alkaline phosphatase activity in undemineralized sections of the rat tibial diaphysis.

Abstract: The distributionofacidand alkaline phosphatase activity in the rat tibia has been studied intransversesectionssawed from freshundemineralized diaphyses.The endostealresorbingsurfaceshad thehighestacidphosphatase activityas demonstrated with50 mMa-naphtholphosphate as the substrate.Strong activitywas present not only in osteoclastsand adjacent osteocytes but extracellularly lining Howship’s lacunae. In areas of bone formation, moderate acid phosphatase activity was present in osteoblasts and young osteocytes. In addition, a zone of activity was found at the mineralizing front where mineralization is initiated. This active zone (a) was separated from the active periosteal cell layer by a zone of inactive osteoid and (b) in formol-calcium-fixed demineralized sections extended into young bone. The identity of the mineralizing front was confirmed by tetracycline labeling. The activity at the mineralizing front had the properties of an enzyme. Alkaline phosphatase activity demonstrated with naphthol ASTR phosphate was largely associated with the osteoblasts and other mesenchymal cells at forming surfaces.

Evaluation of a New System For the Kinetic Measurement of Serum Alkaline Phosphatase

Abstract: Data are presented from the study of a new systematized, kinetic method for the determination of alkaline phosphatase* which was run concurrently in three geographically separated laboratories. The data include estimates of within-day and day-to-day precision, and laboratory-to-laboratory reproducibility. Comparisons are made with three other alkaline phosphatase procedures. An estimated range of normal values is given for adult males and females.

Developmental Regulation of Alkaline Phosphatase in Dictyostelium discoideum

Abstract: The kinetics of accumulation of alkaline phosphatase activity were determined in cells of wild-type and morphologically aberrant mutant strains of Dictyostelium discoideum induced to develop synchronously on membrane supports. The enzyme specific activity increased slowly in wild-type cells until culmination when a dramatic rise in specific activity occurred. The patterns of accumulation in the mutant strains, as well as previous electrophoretic analysis, suggest that the two phases of accumulation may result from the synthesis of distinct isozymes. The rapid accumulation of alkaline phosphatase was found to require concomitant protein synthesis. Ribonucleic acid synthesis, on the other hand, could be inhibited during the 8 hr immediately preceding culmination without affecting the amount of enzyme accumulated. When ribonucleic acid synthesis was inhibited earlier in development, the accumulation of alkaline phosphatase was reduced. Comparison of these results with work on other developmentally controlled enzymes suggests that both transcriptional and translational control occurs during development of D. discoideum. The accumulation of alkaline phosphatase was shown to require specific cellular topography during culmination, suggesting that intercellular interactions which allow synthesis of alkaline phosphatase occur at that stage.

Experimental study of devascularization of the colon.

Abstract: Recent clinical experience (Boley, Schwartz, Lash, and Sternhill, 1963; McGovern and Goulston, 1965; Marston, Pheils, Thomas, and Morson, 1966; Miller and Knox, 1966; Sturdy, 1968) has suggested that many cases of hitherto obscure colonic disease may in fact originate from vascular insufficiency. Some of these cases are directly attributable to blockage of a major vessel by thrombosis or embolism (Marston et al, 1966; Miller and Knox, 1966) whilst in others the ischaemia originates from occlusion or spasm of the smaller intramural vessels (Boley, Krieger, Schultz, Robinson, Siew, Allen, and Schwartz, 1965). The diagnosis of colonic infarction is not always easy and the clinical features, x-ray appearances, and pathology are still not completely defined. The purpose of the present work has been to produce a number of standardized controlled vascular occlusions in the experimental animal and to study their general and local effects in the hope of increasing our knowledge of what may be expected in the clinical situation. Dogs were selected for the investigation for reasons of availability and ease of handling, and also because there is considerable laboratory experience in the behaviour of the ischaemic intestine in this species (Marston, 1962). The pattern of arterial supply to the dog's colon is shown in Figure 1. Blood arrives through the common colic artery, a branch of the superior mesenteric trunk, and the caudal (inferior) mesenteric artery. The common colic artery divides into the middle colic artery, which is distributed much as in the human colon, and the ileo-caeco-colic artery, which runs down the ascending colon and divides into two vessels which finally inosculate with the termination of the superior mesenteric. The caudal mesenteric artery runs straight to the colon and forms a 'T-junction', which above connects with the left branch of the middle colic, and below continues to form the superior rectal artery. In this way a continuous vessel, running along the inner side of the colonic loop, is formed: the marginal artery of the colon. FIG. 1. Anatomy of the blood supply to the dog's colon, showing points of ligature.

Enzymatic and chromosomal characterization of HeLa variants.

Abstract: Seven strains of HeLa cells have been characterized by the number of chromosomes and the activity of the enzymes alkaline phosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, and lactic dehydrogenase. All seven strains were found to differ as to chromosome numbers and enzyme levels despite the fact that two strains were called HeLa and three were called HeLa S3. Three strains were found to have a stemline in which greater than 60% of the cells demonstrated a single chromosome number, and this characteristic was stable for at least 6 months. A nomenclature for these clones has been suggested by the use of the stemline chromosome number as a subscript following HeLa. These three clones were, therefore, designated HeLa65, HeLa71, and HeLa75. Karyotypes were made of the stemlines of these clones and were compared with enzyme levels. Alkaline phosphatase showed the greatest variation from cell line to cell line with a 200-fold difference in levels, whereas glucose-6-phosphate dehydrogenase showed variation in activity over a 12-fold range, lactic dehydrogenase over an 8-fold range, and 6-phosphogluconic dehydrogenase over a 2-fold range. It is suggested that human cell strains can be used for biochemical studies if they are cloned and if the clones are relatively stable at least with respect to modal chromosome number and karyotype.

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

Abstract: 1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme-phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22 degrees the rate constant is 167sec.(-1) for the rearrangement of the initially formed binary complex and is 18sec.(-1) for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme-substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme-phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.

Peptide hydrolase activities of the mucosa of human small intestine

Abstract: Few studies have been published on peptide hydrolase activities of human small intestine mucosa. We developed methods to screen tissue extracts for such enzymes and to quantitate hydrolase activities for dipeptides containing the aromatic amino acid L-phenylalanine. The screening procedure indicated glycyl-L-proline hydrolase activity was reduced in biopsy specimens from patients with flattened intestinal mucosa. To explore this further, we established optimal assay conditions for hydrolase activities (a) glycyl-L-proline, (b) L-phenylalanyl-L-proline, (c) L-alanyl-L-phenylalanine, and (d) L-phenylalanylglycine. Biopsy specimens from patients with various intestinal disorders, but without flattened mucosa, and from three patients with flattened mucosa, showed a disproportionate reduction in activities (a) and (b), with the reduction being significantly more marked in the latter patients. We suggest that intestinal imidopeptide hydrolase activities, such as (a) and (b), are sensitive to changes in intestinal disease generally, particularly to the altered physiology associated with flattening of the mucosa, and are secondary to, rather than a cause of, the intestinal pathology. Our finding that intestinal alkaline phosphatase activity tended to parallel imidopeptide hydrolase activity, and that activity (a) was partially localized to the particulate fraction of mucosal homogenate, suggested that imidopeptide hydrolase activities may be located in the microvilli of the intestinal epithelium and that, like alkaline phosphatase activity, they may be reduced in flattened mucosae, in part at least because of the pathologic changes in the microvilli. In our studies of control subjects we did not detect peptide hydrolase activity deficiency analogous to asymptomatic disaccharidase deficiency.

Localization of virus antigens by enzyme-labelled antibodies.

Abstract: Summary Three enzymes—horseradish peroxidase, alkaline phosphatase, and glucose oxidase—were used as markers in antibody conjugates employed for the detection in the light microscope of either or both T antigens and structural antigens of three viruses: SV40, adenovirus 12 and rat K virus, in infected cell cultures. The specificity of reaction and localization of the antigens was the same with each enzyme and identical with those revealed by fluorochromeconjugates. By using enzymes with reaction products of different colours, two antigens were revealed simultaneously in a single preparation.

Immunological detection of single amino acid substitutions in alkaline phosphatase.

Abstract: Antiserums to wild-type alkaline phosphatase from Escherichia coli were prepared and tested for reactivity with phosphatases altered by point mutations. Eight out of the nine mutant enzymes were distinguished from the wild type with quantitative microcomplement fixation. The structural changes are among the smallest yet observed immunologically.

Cell cycle events in the hydrocortisone regulation of alkaline phosphatase in hela s3 cells

Abstract: The increase in alkaline phosphatase in asynchronous cultures of HeLa S3 cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[3H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.

Production of fetal-like alkaline phosphatase by HeLa cells.

Abstract: Alkaline phosphatase produced by HeLa cells differs in its chemical and physical properties from the enzyme found in adult organs and tissues (Cox and Griffin, 1967). In the present study HeLa cell alkaline phosphatase was compared to a fetal form of the enzyme found in human placenta. Both enzymes have approximately the same molecular weight as judged by sucrose density gradients, and the chemical and physical properties of these alkaline phosphatases are similar. The electrophoretic pattern of the HeLa cell enzyme resembles the placental alkaline phosphatase of the heterozygous FS phenotype except that it is slower moving. Double immunodiffusion using an antibody against HeLa cell alkaline phosphatase and placental and HeLa cell enzymes as antigens shows a single line of partial identity between the two enzymes, with a small spur suggesting additional antigenic sites on the HeLa cell enzyme. The data suggest that malignant cells in culture, HeLa, are producing a fetal-like alkaline phosphatase probably by “derepression of the genome.” However, the electrophoretic and immunological characteristics of the enzyme are altered sufficiently so that it can be distinguished from the normally produced fetal enzyme.

Application of a continuous spectrophotometric assay for 5'nucleotidase activity in normal subjects and patients with liver and bone disease.

Abstract: Serum 5'nucleotidase activity has been measured by a coupled kinetic assay in which adenosine formed by hydrolysis of adenosine 5'-monophosphate in the presence of 150-fold excess of β-glycerophosphate is converted to inosine by adenosine deaminase, with a consequent decrease in absorbance at 265 nm. The method gives activity in proportion to enzyme concentration so long as the rate of decrease of absorbance at 265 nm does not exceed 0.025/min, and not more than 30% of the substrate is consumed. The normal range established in 517 healthy adults was 0-15 mU/ml. A significant correlation between enzyme activity and age was found in females but not in males. Raised levels of 5'nucleotidase activity were found in 92% of patients with obstructive jaundice, 70% of patients with parenchymal liver disease, 81% of patients with hepatic metastases, and 11% of patients with bone disease. The estimation is useful in aiding the elucidation of raised serum alkaline phosphatase activity, and is of value as a liver function test, but is not as frequently increased as alkaline phosphatase in all classes of hepatobiliary disease.

The Specific Binding of Zinc(II) to Alkaline Phosphatase of Escherichia coli

Abstract: The binding of Zn2+ to Escherichia coli alkaline phosphatase has been studied by pH titrations and equilibrium dialysis using 1,10-phenanthroline as competing chelating agent. Measurements of the strength of binding under different experimental conditions have been performed: 1 At constant pH with different concentrations of chelating agent. 2 At different pH with constant concentration of chelating agent. 3 In the presence of denaturing agent at pH 8.0. It was found on the basis of equilibrium dialysis experiments that the binding of the two zinc atoms to alkaline phosphatase may be described as coordination to the two equivalent and independent sites. The high values of the binding constants, and the fact that denaturation destroys the specific binding of zinc to alkaline phosphatase indicates chelate formation with the protein, unless some unknown prosthetic groups are involved.

Enzymes of protein and phosphate catabolism in rat bone. I. Enzyme properties in normal rats.

Abstract: A study was made of several enzymatic activities in rat femur and tibia. Quantitative assays were developed for the determination of acid and alkaline phosphatase, lactate dehydrogenase, proteolytic activity, leucine aminopeptidase, glycylglycine dipeptidase, glutamate dehydrogenase, aspartate transaminase, and alanine transaminase. Some properties of these enzymes were studied using metaphyseal homogenates. The distribution of activity in the metaphysis, diaphysis, and marrow was determined. The activity per mg of DNA was highest in the metaphysis and lowest in marrow. The activities in general were 40–70% lower in the diaphysis than in the metaphysis. Metaphyseal enzymatic activity was the same in 29- and 49-day-old rats but lower in 82-day-old rats. Proteolytic activity at pH 8.0 differed from the other activities studied being higher in marrow than in bone and increasing markedly with age.