Plasma Membrane Enzyme Activities Research Articles

Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents.

The development of safe antimicrobial agents is important for the effective treatment of pathogens. From a multitude of discovered inhibitory compounds, only a few antimicrobial agents are able to enter the market. Many antimicrobials are, on the one hand, quite effective in killing pathogens but, on the other hand, cytotoxic to eukaryotic cells. Cell health can be monitored by various methods. Plasma membrane integrity, DNA synthesis, enzyme activity, and reducing conditions within the cell are known indicators of cell viability and cell death. For a comprehensive overview, methods to analyze cytotoxic and hemolytic effects, e.g., lactate dehydrogenase release, cell proliferation analysis, cell viability analysis based on the activity of different intracellular enzymes, and hemolysis assay of antimicrobial compounds on human cells, are described in this updated chapter.

Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents.

The development of safe antimicrobial agents is important for the effective treatment of pathogens. From a multitude of discovered inhibitory compounds only few antimicrobial agents are able to enter the market. Many antimicrobials are, on the one hand, quite effective in killing pathogens but, on the other hand, cytotoxic to eukaryotic cells. Cell health can be monitored by various methods. Plasma membrane integrity, DNA synthesis, enzyme activity, and reducing conditions within the cell are known indicators of cell viability and cell death. For a comprehensive overview, methods to analyze cytotoxic and hemolytic effects, e.g., lactate dehydrogenase release, cell proliferation analysis, cell viability analysis, and hemolysis assay of antimicrobial compounds on human cells, are described in this chapter.

Activation of plasmalemmal NADPH oxidase in etiolated maize seedlings exposed to chilling temperatures

Five-day-old etiolated seedlings of maize (Zea mays L.) were used to study the kinetics of hydrogen peroxide formation upon lowering growth temperature from 25 to 6°C. The total content of hydrogen peroxide in root and shoot tissues increased by 30–40% after 2-h cooling compared to the control level but returned to the initial level or decreased even lower after 24-h cooling. In order to prove the involvement of plasma membrane NADPH oxidase in changes of hydrogen peroxide content upon cooling, isolated plasma membranes were obtained from untreated plants and from seedlings chilled at 6°C for 2 and 24 h. The NADPH-dependent generation of superoxide anion radical in isolated plasma membranes was quantified by measuring the rate of formazan production from the tetrazolium salt XTT. The activity of plasma membrane NADPH oxidase in shoots was 50 ± 9 nmol O2/(mg protein min), which was 1.5 times higher than the activity in roots. The enzyme activity in plasma membranes was inhibited by low concentrations of diphenyleneiodonium. The effective concentration EC50 was 5.10 μM for shoots and 9.05 μM for roots. The activity of plasma membrane NADPH oxidase increased after 2-h cooling of seedlings but reversed to the control level after 24-h cooling. This transient activation of NADPH oxidase upon cooling was similar to the pattern of hydrogen peroxide formation in shoots and roots. Analysis of NADPH oxidase activity of plasma membrane proteins after their separation in denaturing conditions followed by subsequent renaturation revealed four diphenyleneiodonium-sensitive bands with mol wt of 130, 88, 51, and 48 kD. Western blot analysis of the reaction with antibodies against the catalytic domain of phagocyte NADPH oxidase revealed the proteins with mol wt of only 88 and 48 kD. The properties of molecular organization of plasma membrane NADPH oxidase are discussed in terms of its role in cell signaling.

The metabolic activity of neutrophils and monocytes as the model for the study of the organism biocompatibility with different materials

In stimulated status neutrophils and monocytes in the foci of introduction the different materials are capable to excrete of biologically active substances by means of which if influence on processes of reparation. The express changes of blood leukocytes activity in response on the introduction of stimulated agents are assume as a basis of the proposed model of the organism biocompatibility with different materials. On example of the influence of different materials (ceramics, titanium, latex, surgical steel and copper), from which can be made implant and surgical instruments it was showed that the leukocytes were showed the different reaction of enzyme systems. This model allows the completely and objectively to study the influence of different materials on the neutrophyls and monocytes--the effected cells of the inflammatory process. Moreover, the activity of cation proteins and plasma membrane enzymes of leukocytes is the most expressed factor.

Fluorescence Spectroscopic Studies of Pressure Effects on Na+,K+-ATPase Reconstituted into Phospholipid Bilayers and Model Raft Mixtures

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.

Translocation of lysophosphatidic acid phosphatase in response to gonadotropin-releasing hormone to the plasma membrane in ovarian cancer cell

Objective Lysophosphatidic acid mediates proliferative and/or morphologic effects on multiple cell lineages, which include ovarian cancer cells. Lysophosphatidic acid hydrolysis limits the duration of lysophosphatidic acid's action. We examined hormonal translocation of lipid phosphate phosphatase type 3 to the plasma membrane in gonadotropin-releasing hormone–responsive ovarian cancers. Study design Ovarian cancers that were removed surgically and the ovarian cancer cell lines Caov-3 and SK-OV-3 were examined. Lipid phosphate phosphatase type 3 protein and activity in plasma membranes were assessed by immunohistochemical staining with lipid phosphate phosphatase type 3–specific antibodies and by the measurement of the conversion of exogenous [ 3H-oleoyl]lysophosphatidic acid to mono[ 3H-oleoyl]glycerol, respectively. Results In ovarian cancers that were removed surgically, the cell surface staining and activity measurements indicated that a portion of the enzyme was localized to the plasma membrane. In Caov-3 cells and SK-OV-3 cells, lipid phosphate phosphatase type 3 protein was present both in the cytoplasm and at the plasma membrane. Treatment of the cells with a gonadotropin-releasing hormone agonist buserelin produced a rapid and progressive translocation of lipid phosphate phosphatase type 3 protein to the plasma membrane, with a concomitant loss of cytoplasmic staining. The enzyme activity in plasma membrane was also increased when the cell lines were exposed to the gonadotropin-releasing hormone agonist in intact cells before the assay of the cell membranes. Conclusion These findings support the presence of lipid phosphate phosphatase type 3 in plasma membrane of ovarian cancers and provide for the ability of agonists (such as gonadotropin-releasing hormone) to induce the translocation of lipid phosphate phosphatase type 3 to plasma membrane in ovarian cancer cells.

Influence of Estrogen Cytostatics on Activity of Plasma Membrane Enzymes 5'-Nucleotidase and N+-K+-ATPase

We studied the effect of four conjugated synthetic derivatives of estrone and ethynylestradiol and bis-beta-chloroethylamine-containing substance on activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase. As differentiated from precursors, estrogen cytostatics decreased activity of plasma membrane enzymes. Reference preparations chlorophenacyl and estradiol had little effect on activity of 5'-nucleotidase and N+-K+-ATPase. These data suggest that damage to plasma membrane enzymes is related to the effect of estrogen cytostatic molecules. Test compounds produced an antiproliferative effect on estrogen-independent tumor cells, which strongly correlated with a decrease in activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase. The derivative of ethynylestradiol with the cytostatic residue in the 3-position of the steroid nucleus (Po-714-11alpha) most significantly modulated enzyme activity.

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment.

The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1:4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

Effect of Acetaminophen on Na+, K+ ATPase and Alkaline Phosphatase on Plasma Membranes of Renal Proximal Tubules

In previous work we reported that 1 h after acetaminophen (APAP) administration, tubular function remained at control values, while 16 h later a significant deterioration of tubular function was observed. The aim of the present work was to study if APAP induces its renal toxic effects by altering the normal activity of key tubular plasma membrane enzymes. We analyzed the effects of a nephrotoxic dose of APAP (1000 mg/kg b.wt., i.p.) on the activities of the brush-border membrane (BBM) enzyme, alkaline phosphatase, and the basolateral membrane (BLM) enzyme Na+, K+ ATPase 1 h (APAP1h) and 16 h (APAP16h) after dosing. Na+, K+ ATPase abundance in homogenates and each membrane domain were analyzed by Western blot. Cortical adenosine 5′ triphosphate (ATP) content was also evaluated. At each time studied, APAP promoted a diminution of alkaline phosphatase in BBM. Na+, K+ ATPase activity in BLM showed a biphasic response to APAP. One hour after APAP administration it was significantly increased, but it was decreased 16 h after dosing. Na+, K+ ATPase protein abundance was elevated in homogenates, BLM, and BBM after 1 h of APAP dosing. After 16 h, Na+, K+ ATPase abundance was increased in homogenates, while in BLM it was decreased. No differences were observed in cortical ATP content in each time studied. Our present results could contribute to the understanding of the molecular basis of the previously reported time course alteration in the fractional excretion of sodium promoted by a nephrotoxic dose of APAP.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans.

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

24,25-(OH)2D3 regulation of matrix vesicle protein kinase C occurs both during biosynthesis and in the extracellular matrix.

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCalpha) and PKCzeta, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)2D3 [14]. In the present study, we examined whether the effect of 24,25-(OH)2D3 on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10(-9)-10(-7) M 24,25-(OH)2D3. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)2D3-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24, 25-(OH)2D3-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)2D3-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)2D3 on PKC involved phospholipase A2 (PA2). Quinacrine (a PA2 inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)2D3, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA2 plays a role in the nongenomic regulation of PKC by 24,25-(OH)2D3. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)2D3 modulated the distribution of PKCalpha, beta, and zeta between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24, 25-(OH)2D3 regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation.

Effect of colchicine and trimethylcolchicinic acid on CCl4-induced cirrhosis in the rat.

Colchicine is one of the most promising drugs for the treatment of cirrhosis. However, due to its toxicity, other drugs are being evaluated and colchicine-like molecules may be good alternatives. The aim of this work was to compare the beneficial effects of colchicine and trimethylcolchicinic acid (a colchicinoid less toxic than colchicine) on CCl4-cirrhosis. The drugs were administered either through CCl4 administration (8 weeks) or after CCl4 intoxication for 4 weeks at a dose of 10 micrograms/rat/day, orally. Liver plasma membranes were isolated for high affinity Ca(2+)-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase activities. The activities of gamma-glutamyl transpeptidase and alkaline phosphatase were also measured in serum. Liver glycogen content and a marker for lipid peroxidation were determined in liver samples. We found that both compounds preserved and significantly reversed high affinity Ca(2+)-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase plasma membrane and serum enzyme activities as well as the hepatic glycogen content.

Vitamin D regulation of metalloproteinase activity in matrix vesicles.

Matrix vesicles (MVs) are enriched in matrix metalloproteinases (MMPs) capable of degrading proteoglycans. The aim of the present study was to identify which MMPs are present in MVs and determine whether these MMPs are regulated by 1,25-(OH)2D3 [1,25] and 24,25-(OH)2D3 [24,25]. To do this, growth zone (GC) and resting zone (RC) chondrocytes were isolated from rate costochondral cartilage and placed into culture. At confluence, GCs were treated with 1,25 and RCs with 24,25 for 24 hours. MVs, plasma membranes (PMs), and conditioned media were then collected from the cultures. RTPCR demonstrated the presence of mRNA for stromelysin-1 and 72 kDa gelatinase in both RCs and GCs, Casein zymography revealed activity at M(r) 48 and 28 kDa in MV, but not PM or conditioned media; Western analysis confirmed that this activity was stromelysin-1. Gelatinolytic activity, at low levels, was also found in MVs, but not PMs or conditioned media. When enzyme activity was measured using a proteoglycan bead assay, it was found that both GCs and RCs produced MVs and PMs containing neutral metalloproteinase. Both cells also produced MVs and PMs containing plasminogen activator. The addition of 1,25 to GCs caused a significant 4- to 5-fold increase in metalloproteinase activity in MVs, but not PMs. In contrast, MVs from cultures of RCs treated with 24,25 contained decreased metalloproteinase activity; enzyme activity in PMs was unaffected by 24,25. Plasminogen activator in MVs from RC was increased by treatment with 24,25, while MV enzyme activity was decreased after treatment of GC cultures with 1,25. This study shows that both RCs and GCs produce stromelysin-1 and 72 kDa gelatinase and that these enzymes are preferentially localized in MVs. Further, MMP and plasminogen activator activities in MVs and PMs are regulated by vitamin D metabolites.

Acetylcholinesterase activity of synaptic plasma membranes during ageing: effect of L-acetylcarnitine.

A physiopathological role for acetylcholine (ACh) was hypothesized during ageing and related neurodegenerative diseases, e.g. dementia. This research was aimed to study acetylcholinesterase (AChE) activity during development and ageing of the frontal cerebral cortex of 4-, 8-, 12-, 16-, 20- and 24-month-old rats. This study was performed on synaptic plasma membranes, the specific subcellular compartment where the enzyme is located in vivo both in control animals and after in vivo acute treatment with L-acetylcarnitine. Maximum AChE activity was unaffected by age, and L-acetylcarnitine treatment increased enzyme activity in synaptic plasma membranes of 8-month-old rats. A comprehensive analysis of these results suggests: (a) the observed alterations in protein can substantially affect neurochemical data if results are presented as specific activities per unit protein; (b) energy metabolism plays the major role in the disturbed ACh metabolism during ageing and (c) the understanding of the mode of action of L-acetylcarnitine in treatment of dementia.

Localisation of the multidrug resistance-associated protein, MRP, in resistant large-cell lung tumour cells

The drug transport protein, P-glycoprotein, confers multidrug resistance (MDR) by expelling drugs across the cell surface. The structurally similar multidrug resistance-associated protein, or MRP, is also involved with drug efflux. In MDR variants of the human lung tumour cell line COR-L23 that overexpress MRP, there are also changes in intracellular drug distribution. To ascertain whether MRP could be involved in either process, experiments were performed to identify where MRP was located in these cells. Following separation of membranes by sucrose gradient centrifugation, MRP was found predominantly in the lighter membrane fractions containing plasma membrane enzyme activity. Immunofluorescent staining with a monoclonal antibody raised against MRP confirmed that MRP is present at the cell surface of these MDR lung tumour cells.

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na K - ATPase activity was 65.9 ± 18.7 and 38.6 ± 22.8 ( P < 0.001) nmol/mg protein × min for control and high-expression transfected cells, respectively. Consistent with this observation, [ 3H] ouabain binding to whole cells was significantly decreased from 3.7 ± 0.3 to 2.0 ± 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na +, K +-ATPase protein; in contrast, the activities of 5′-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na K - ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na K - ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.

Effect of Castration and Anatomical Site on the Plasma Membrane Structure and the Activities of Adenylate Cyclase and 5'-Nucleotidase in Pig Adipocytes

The effects of castration on fat cell plasma membrane structure and enzyme activities (adenylate cyclase and 5'-nucleotidase) were studied in pig adipose tissues in two fat deposits (subcutaneous and perirenal). Castration induced a fat cell enlargement in both tissues. Membrane cholesterol content was reduced and fluidity was increased in perirenal fat from castrated animals. Castration had no effect on 5'-nucleotidase activity which was higher in subcutaneous than in perirenal in both kinds of animals. Adenylate cyclase activity was studied in the presence of different effectors: isoproterenol-stimulations of the enzyme were not affected by castration but were site-specific. GppNHp-stimulated activities were increased in subcutaneous fat from castrated animals. Castration had no influence on forskolin stimulations. The magnitude of GppNHp- and forskolin-stimulated activities were found to be tissue-dependent. Membrane results are discussed in relation with castration-induced fat cell enlargement.

Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures.

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 microM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 microM A23187 in resting zone cells and at 0.1 and 1 microM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity is isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.

Senescence of oat leaves: changes in the free sterol composition and enzyme activities of the plasma membrane

Plasma membrane fractions were prepared from young (10-day-old), mature (17-day-old) and senescent (10 + 7-day-old) oat leaves ( Avena sativa L.) through aqueous two-phase partitioning and analyzed for their free sterol composition. Continued leaf growth resulted in a strong increase in overall sterol content from 40 nmol · mg −1 lipid to about 120 nmol · mg −1 lipid (1.5% and 5% by wt. of total lipids respectively). This increase could be completely ascribed to increased cholesterol and stigmasterol concentrations. Kinetic parameters of vanadate-sensitive ATPase and NADH-FeCN reductase activities did not change significantly with leaf maturation. The sterol concentration increased to a lesser extent (4.2% and 4.4% by weight) in the plasma membrane of 10-day-old leaf segments that were cut and kept in light or dark for 7 more days. However, the increase in this case was completely due to a raise in Δ 7-avenasterol and 28-isofucosterol concentrations. The K m and V max values for the ATPase activity decreased significantly in these membranes. The characteristics of the FeCN 2 reductase and of glucan synthetase II activity were apparently not affected. The effect of senescence on sterol biosynthesis and on plasma membrane enzyme activities is discussed.