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R A Stinson

Publications and source records attributed to R A Stinson.

At least 19 recordsLinked to original sources
PubMed2001

The continuing professional development of the Canadian Society of Clinical Chemists and the Canadian Academy of Clinical Biochemists.

The Canadian Society of Clinical Chemists (CSCC) and the Canadian Academy of Clinical Biochemistry (CACB) have recently implemented a new professional development program for its 400 members. The program's goals are: to evaluate and recognize professional development based on self-determined needs, interests, and learning preferences; and to ensure that qualified professionals directing clinical biochemistry laboratories have adequate basic and current knowledge to function competently in their profession. Involvement in the program is currently voluntary and based on a 3-year cycle during which time participants must earn a minimum of 150 credits from at least 3 of 8 categories' learning activities. Of these activities: four are related to updating knowledge (Formal Group Learning related to Laboratory Medicine, Other Formal Group Learning, Self-Directed Learning, Self-Assessment); three are related to the maintenance and implementation of practice skills (Service Associated Learning, Teaching, Change in Practice); and one is related to the advancement of knowledge (Publications and Presentations). One credit is defined as one hour of continuing professional development activity. At the end of each year, members document their activities by submitting a 4 page Annual Summary of Activities (ASA) form. The cost of coordinating the program is minimal as it is administered by a steering committee and smaller working committees, all of whom are voluntary. A basic assumption of our program is that self-management of professional development (PD) is an important prerequisite and indicator of maintenance of competence. By recognizing learning through a number of activities and outcomes, it is anticipated that our program will promote an overall improvement in the quality of Laboratory Medicine throughout Canada.

Canada↗
PubMed1994

Human liver plasma membranes contain an enzyme activity that removes membrane anchor from alkaline phosphatase and converts it to a plasma-like form.

Treatment of liver plasma membranes with Triton X-100 allowed an endogenous alkaline phosphatase-converting activity to convert amphiphilic alkaline phosphatase (membrane anchor covalently attached) to hydrophilic dimers that resemble the enzyme found in normal plasma. The Triton-solubilized activity was unaffected by protease inhibitors. Amphiphilic alkaline phosphatase purified from human liver and placenta were both substrates. The Triton-solubilized enzyme would not hydrolyze L-3-phosphatidyl(2-3H)-inositol or p-nitrophenylphosphoryl choline, nor would it cleave endogenous alkaline phosphatase from intact plasma membranes. These observations and the analysis of the protein product of the hydrolysis of placental alkaline phosphatase, following treatment with the converting activity, indicated that the enzyme has the specificity of a glycosyl-phosphatidylinositol phospholipase D. Further characterization of the enzyme activity suggests additional similarities with the glycosyl-phosphatidylinositol phospholipase D found in mammalian plasma. Alkaline phosphatase-converting activity in plasma membranes represented the same percent of total protein as it did in whole liver, whereas serum contained 3- to 10-times this amount. Endogenous converting activity in plasma membranes was not solubilized by salt washes, sonication, or repeated freeze-thaw treatments. We believe it is unlikely that the alkaline phosphatase-converting activity in liver plasma membranes resulted from adsorption of the enzyme present in plasma.

Alkaline Phosphatase↗
PubMed1993

Expression and nature of the alkaline phosphatase gene in cultured osteosarcoma cells.

The molecular mechanism for the differences in specific activity of alkaline phosphatase in six human osteosarcoma cell lines was investigated. Five of the lines expressed only the tissue-non-specific or liver/bone/kidney isoenzyme of alkaline phosphatase. The sixth line had the lowest levels of alkaline phosphatase and this was determined to be a mixture of liver/bone/kidney isoenzyme and at least one other form. The mRNA of liver/bone/kidney alkaline phosphatase was identified by Northern analysis in the three cell lines that expressed the largest amount of alkaline phosphatase catalytic activity. This mRNA was indistinguishable in size from that seen in control mRNA from normal kidney (2.5 kb). Southern analysis demonstrated that EcoRI or HindIII restriction fragment patterns and the intensity of the bands, of the liver/bone/kidney alkaline phosphatase gene in the osteosarcoma cell lines were identical to that of the control DNA from normal peripheral blood leukocytes. Thus, the gene coding for liver/bone/kidney alkaline phosphatase appears to be intact in all of these osteosarcoma cells and it is unlikely that rearrangement, deletion or amplification of the gene is responsible for its activation or inactivation. Slot blot analysis revealed varying amounts of the transcripts of the liver/bone/kidney isoenzyme in each of the cell lines. The best fit line of a plot of the log of the level of mRNA of alkaline phosphatase vs. the log of the specific activity of liver/bone/kidney alkaline phosphatase was constructed. This gave a Pearson correlation coefficient of 0.92 (P < 0.008), demonstrating a significant relationship between the two variables. It is likely that the regulation of alkaline phosphatase activity is at the transcriptional process rather than the translational or post-translational processes and that the specific activity of the enzyme may be controlled by the amount of steady-state mRNA of the liver/bone/kidney isoenzyme.

Alkaline Phosphatase↗
PubMed1993

Characterization of the alkaline phosphatase expressed on the surface of a Hodgkin's lymphoma cell line.

Alkaline phosphatase solubilized from a human Hodgkin's lymphoma cell line (L428) was compared with purified amphiphilic and hydrophilic forms of the enzyme from human liver, and with the enzyme solubilized from a cultured osteosarcoma cell line (Saos-2). Purified hydrophilic alkaline phosphatases from human placenta and intestine were also compared in some experiments. Alkaline phosphatase was released from the plasma membrane of intact lymphocytes by phosphatidylinositol phospholipase C and thus is anchored to the outside of the plasma membrane by covalently attached phosphatidylinositol. Enzyme released in this way was hydrophilic and that solubilized with Triton X-100 was amphiphilic, as assessed by adsorption to octyl-Sepharose. Lymphocyte alkaline phosphatase, when released from the membrane by phosphatidylinositol phospholipase C or solubilized by Triton X-100, had apparent M(r) values on gradient gel electrophoresis of 227 and 494 kDa, respectively. These values were consistently higher than equivalent ones obtained with enzymes purified from human liver, but were similar to those of cultured osteosarcoma cells. Isoenzyme-specific inhibitors of alkaline phosphatase showed similar patterns of inhibition between the enzyme from L428 cells and the tissue-nonspecific (liver/kidney/bone) isoenzyme from human liver. Heat stabilities were similar for the enzymes from L428 and Saos-2 (bone isoform) cell lines, but differed significantly from those of liver, intestine and placenta. We conclude that the alkaline phosphatase expressed in this lymphoma cell line (L428) has properties that most closely resemble those of the tissue-nonspecific isoenzyme found normally in osteoblasts of bone (bone isoform).

Alkaline Phosphatase↗
PubMed1993

Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis.

Nine different isoenzymes and (or) isoforms of alkaline phosphatase (ALP; EC 3.1.3.1) from human tissue were studied with respect to Km and Vmax values for p-nitrophenyl phosphate (p-NPP) in seven different potential phosphoacceptors/buffers. Generally, the phosphoacceptors/buffers with the lowest affinity for p-NPP (highest Km values) gave the highest Vmax values; for the nine enzyme forms in this study, the mean Km and Vmax values were greatest in 2-(ethylamino) (EAE). The two amino-propanol buffers gave the lowest Km and Vmax values. The phosphoacceptors/buffers N-methyl-D-glucamine (MEG), diethanolamine, and Tris had intermediate Km and Vmax values. Hydrophilic liver ALP retained > 90% of its activity after 24 h at 30 degrees C in both 1.0 and 0.3 mol/L Tris and 2-amino-2-methyl-1,3-propanediol and in 0.3 mol/L MEG. This isoenzyme showed greatest inactivation upon prolonged exposure to 1.0 and 0.3 mol/L EAE, the activity at 24 h being approximately 50-66% of that at zero time. p-NPP underwent the greatest spontaneous degradation, approximately 2.5 times that of baseline levels, in 1 mol/L MEG. There was little degradation in all of the buffers tested at 0.3 mol/L or in Tris, EAE, and 2-amino-2-methyl-1-propanol at 1.0 mol/L.

Alkaline Phosphatase↗
PubMed1991

High-molecular-weight alkaline phosphatase in serum has properties similar to the enzyme in plasma membranes of the liver.

Partially purified high-molecular-weight alkaline phosphatase from serum was compared with two other forms of the enzyme from the human liver, enzyme in native plasma membranes and purified alkaline phosphatase as a hydrophilic dimer. In a high-molecular-weight form from serum and plasma membranes, and when treated with 1% (v/v) Triton X-100, alkaline phosphatase showed a major band on gradient gel electrophoresis with a mobility equivalent to 400 kD. Nondetergent-treated material from both sources did not enter the gel and was in the voided volume of a gel permeation column. Stimulation of catalytic activity by four different phospholipids and by albumin yielded similar results for high-molecular-weight alkaline phosphatase and for the enzyme in plasma membranes, but these were different from the hydrophilic form. Inhibitors of alkaline phosphatase had similar effects on all forms. Of the three forms of the enzyme, only the hydrophilic dimer did not become incorporated into liposomes or adsorb to octyl-Sepharose after solubilization with Triton X-100 and removal of the detergent. Km (substrate concentration to give half maximal velocity) values with p-nitrophenylphosphate and heat and sodium dodecyl sulfate stabilities were similar for all forms. In the high-molecular-weight form from serum and in plasma membranes, alkaline phosphatase and 5'-nucleotidase showed similar rates of release by phosphatidylinositol phospholipase C. Three preparations of phospholipase D failed to release alkaline phosphatase from either the high-molecular-weight form or from plasma membranes. Based on these similarities, it is probable that the complex of high-molecular-weight alkaline phosphatase in serum most often originates from fragments of hepatic plasma membranes.

Alkaline Phosphatase↗
PubMed1991

Properties of amphiphilic and hydrophilic forms of alkaline phosphatase from human liver.

Amphiphilic and hydrophilic forms of alkaline phosphatase differed in electrophoretic mobility, sensitivity to heat, activation by phospholipids and albumin, and affinity of monoclonal antibodies, but were similar in substrate Km and inhibitor Ki values, sensitivity to sodium dodecyl sulfate, and electrophoretic behavior on desialylation. Chemical cross-linking experiments failed to conclusively demonstrate an aggregated state of amphiphilic alkaline phosphatase in Triton X-100. Further, attempts to identify a polymeric hybrid between amphiphilic forms of human liver and placental alkaline phosphatase were unsuccessful. We conclude that the covalent attachment of the hydrophobic phosphatidyl-inositol membrane anchor causes the amphiphilic form to behave anomalously on electrophoresis and to affect certain of the enzyme's catalytic and physical properties.

Alkaline Phosphatase↗
PubMed1990

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol.

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.

Alkaline Phosphatase↗
PubMed1989

Biochemical and morphological effects of human hepatic alkaline phosphatase in a neonate with hypophosphatasia.

Enzyme replacement-therapy for a severely affected premature boy (birthweight: 2,380 g, GA: 36 weeks) with hypophosphatasia was attempted by infusions of purified human hepatic alkaline phosphatase. Treatment (1.2 IU/kg/min) started at age three weeks and was repeated in weekly intervals until age 10 weeks, when the child died. Samples of alkaline phosphatase were diluted with 10 ml of physiological saline and infused over 30 min via an umbilical arterial catheter. No toxic or allergic side effects were observed. Serum alkaline phosphatase activity increased from 3 IU/L before treatment to a maximum level of 195 IU/L with a half-life time between 37 and 62 hours. Urinary excretion of phosphoethanolamine decreased during therapy from a maximal level of 9.5 to 5.5 mumol/mg creatinine (normal: less than 0.4 mumol/mg creatinine). Calcium, phosphorus, parathormone and 1,25-diOH vitamin D levels were within normal range. Sequential radiographic studies showed no improvement of bone mineralization. Bone morphology was studied by light and electron microscopy before treatment and post mortem. The borderline between mineralized and unmineralized matrix was more distinct after treatment and on the electron microscopical level initial spots of mineralization were more frequent between the collagen fibrils compared to the biopsy specimen before treatment. In contrast to previous studies however, only woven and bundle bone structures were studied from the tibial crest, where the lack of osteoblast-like cells upon the newly formed osteoid matrix was prominent.

Alkaline Phosphatase↗
PubMed1988

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis.

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.

Alkaline Phosphatase↗
PubMed1988

Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phospholipase C: effect of tunicamycin.

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], EC 3.1.3.1) expressed in two human osteosarcoma cell lines (Saos-2 and KTOO5) in culture was the tissue nonspecific type and was released from the plasma membrane by phosphatidylinositol (PI) phospholipase C. Despite a difference of 10-fold between the two cell lines in the amount of alkaline phosphatase expressed, the phospholipase solubilized nearly all of the phosphatase from resuspended cells of the two lines. Alkaline phosphatase released with Nonidet-P40 from Saos-2 cells had a Mr of 445,000 by gradient gel electrophoresis in the absence of detergent; that released by PI-phospholipase C was 200,000. The subunit Mr of both solubilized forms was 86,000. Thus, tetrameric alkaline phosphatase in the membrane is attached by a PI-glycan moiety and is converted to dimers when released by PI-phospholipase C. Tunicamycin treatment of Saos-2 cells in culture affected the release of alkaline phosphatase by a high concentration of PI-phospholipase C, but not by a low concentration; both the rate and extent of release were lower from treated cells. However, the enzyme released from the treated cells was in two forms with different molecular weights; it seems that both glycosylated and nonglycosylated dimers were transported to the cell surface and incorporated into the plasma membrane. Glycosylation does not appear to be necessary for alkaline phosphatase to be anchored in the membrane via PI.

Alkaline Phosphatase↗
PubMed1987

Phosphotransferase activity of human alkaline phosphatases and the role of enzyme Zn2+.

Purified isoenzymes of human alkaline phosphatase from placenta, intestine and liver were investigated as catalysts for phosphotransferase activity, using the phosphoacceptors Tris, 2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol, diethanolamine, 2-(ethylamino)ethanol, ethanolamine, and N-methyl-D-glucamine. All of the compounds supported phosphotransferase catalysis, conforming to saturation kinetics. There was little difference among the isoenzymes with respect to Km values of the acceptors, but the liver form was the most efficient (highest Vmax/Km) in forming phosphoacceptors; it was also the most efficient (highest Vamax/Ka) when the phosphoacceptors were considered as activators. At Vmax the isoenzymes differed little in their support of phosphotransferase activity relative to phosphohydrolysis, although the intestinal enzyme tended to be the poorest. The two best acceptors were diethanolamine, providing the highest phosphotransferase velocity, and 2-(ethylamino)ethanol, having the lowest Km. The phosphoaceptors that bound Zn2+ tightly did not function well in the phosphotransferase reaction, and vice versa. However, temporal assessment of the phosphohydrolytic and phosphotransferase activities during removal of Zn2+ from the enzyme with 1,10-phenanthroline revealed no evidence of a special role for Zn2+ in the latter activity.

Alkaline Phosphatase↗
PubMed1987

Tetrameric alkaline phosphatase from human liver is converted to dimers by phosphatidylinositol phospholipase C.

Membrane-bound human liver alkaline phosphatase solubilized by a non-ionic detergent, Nonidet P-40 (NP-40), has the molecular mass of a tetramer. It can be converted to a dimeric form by treatment with a phosphatidylinositol phospholipase C (PI-PLC) obtained from Bacillus cereus. When human liver plasma membranes were directly treated with PI-PLC, the released alkaline phosphatase was dimeric. Thus, phosphatidylinositol may help maintain the tetrameric quaternary structure of alkaline phosphatase and aid its binding to human liver plasma membranes.

Alkaline Phosphatase↗
PubMed1987

Isoenzymes of alkaline phosphatase in amniotic fluid: implications in prenatal screening for cystic fibrosis.

Utilizing their differential susceptibilities to inhibitors and heat, we determined the amounts of the placental, liver, and fetal-intestinal isoenzyme forms of alkaline phosphatase in 143 samples of normal amniotic fluid obtained at 14 to 18 weeks' gestation (1). For reliable results, it was necessary to standardize inhibition profiles for each pure isoenzyme in amniotic fluid. Total activity and the absolute amounts of placental and fetal-intestinal activities were significantly related to gestational age (p less than 0.05). These relationships that were absent when activities were expressed as percentages of the total. The mean isoenzyme composition of the 143 samples, expressed as a percentage of total alkaline phosphatase activity, was: placental, 3.4%; liver, 9.8% (maximum, 47%); and fetal intestinal, 87% (minimum, 53%). The presence of phosphate in the assay medium (13.5 mmol/L) profoundly and differentially inhibited the isoenzymes of alkaline phosphatase and changed the inhibition profiles of the tissue-specific enzymes; thus, it would not be feasible to use inhibitors to differentiate the forms. We therefore propose a phosphate-free technique for quantifying the isoenzymes of alkaline phosphatase in amniotic fluid obtained at 14 to 18 weeks' gestation, to achieve the highest predictive values in a prenatal diagnostic test for cystic fibrosis.

Alkaline Phosphatase↗