The distributions of some granule-associated enzymes in guinea-pig polymorphonuclear leucocytes

1. Homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction. 2. The distributions in these fractions of protein, DNA, succinate dehydrogenase, beta-glucuronidase, peroxidase, alkaline phosphatase, acid phosphatase (against p-nitrophenyl phosphate and beta-glycerophosphate), cathepsin, and catalase were compared. 3. Almost all of the DNA sedimented in the first two pellets, indicating that the nuclei were relatively intact. 4. The four hydrolases and peroxidase showed different distribution patterns, although these activities were previously reported to be localized mainly in the single ;granule' fraction isolated from leucocytes. 5. The particles containing peroxidase, acid phosphatase and alkaline phosphatase all exhibited latency. Maximum activity for each enzyme was obtained at roughly similar concentrations of Triton X-100. 6. The acid phosphatase of these cells was distributed between two populations of particles that differed in both sedimentation characteristics and density. The acid phosphatase(s) of the two populations showed slightly different substrate specificities. This bimodal distribution was not an artifact of the procedure used to elicit the cells. 7. Catalase was recovered almost entirely in the soluble fraction and showed no latency in freshly prepared homogenates. No urate oxidase was detected. 8. We conclude that the ;granule' fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.     

Studies on the NADPH oxidizing activity in polymorphonuclear leucocytes: The mode of association with the granule membrane the relationship to myeloperoxidase and the interference of hemoglobin with NADPH oxidase determination

Phospholipase C-treated polymorphonuclear leucocytes were used to study the properties of NADPH oxidase activity of stimulated polymorphonuclear leucocytes.A comparison of the effects of phospholipase C treatment of whole leucocytes on the NADPH oxidase activity with other granule enzymes showed that the activities of β-glucuronidase and acid phosphatase were un-affected, whereas the NADPH oxidase activity was stimulated 4-fold and myeloperoxidase was inhibited about 30%.The distribution of NADPH oxidase activity among subcellular fractions of polymorphonuclear leucocyte homogenates was unaffected by phospholipase C whereas the other enzymes were released into the medium in soluble form; β-glucuronidase > acid phosphatase and myeloperoxidase.A number of solubilizing agents and procedures were tested for their ability to release NADPH oxidase activity from granules of phospholipase C-stimulated polymorphonuclear leucocytes. All procedures used caused appreciable release of granule protein but no release of NADPH oxidase activity. Most of the procedures used strongly inhibited the oxidase activity. These results indicate that the enzyme is tightly bound to granule structures and that the integrity of these structures is required for activity.Some of the solubilizing agents used (KCI, guanidium chloride) were very effective in solubilizing myeloperoxidase.The differential response of myeloperoxidase and NADPH oxidase to treatment with phospholipase C or solubilizing procedures suggests that the two activities are not due to the same enzyme. However, definite conclusion cannot be drawn because of the complex nature of myeloperoxidase.It was found necessary to lyse any erythrocytes present as contaminants of polymorphonuclear leucocytes preparations, since hemoglobin was converted to methemoglobin during the NADPH oxidase assay and methemoglobin exhibits appreciable NADPH oxidase activity.     

Subcellular distribution of glycosidases in human polymorphonuclear leucocytes.

The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the 'tertiary' granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2--38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.     

Two distinct mechanisms for stimulation of oxygen-radical production by polymorphonuclear leucocytes.

Oxygen-radical production stimulated from rat polymorphonuclear leucocytes by either unopsonized latex particles (diameter = 1.01 microM) or chemotactic peptide (N-formyl-Met-Leu-Phe) was monitored by using luminol-dependent chemiluminescence. Azide inhibited by more than 80% the luminescence response induced by chemotactic peptide whether added before or after stimulation. However, the luminescence response to latex particles was progressively less susceptible to azide inhibition if the azide was added after the stimulus. Cytochalasin B, which was shown to abolish phagocytosis of the latex beads, also abolished the chemiluminescence response. However, the same cells showed a greatly enhanced response to chemotactic peptide. Cytochalasin B-treated cells secreted approx. 45% of total cellular myeloperoxidase in response to chemotactic peptide, but there was no detectable secretion in response to unopsonized latex particles. Microperoxidase equivalent to 20% of cellular peroxidase activity added to the cells before addition of the stimulus had no effect on the response to latex particles but increased approx. 2-fold the peak rate of chemiluminescence induced by chemotactic peptide. It was concluded that the unopsonized latex particles stimulated oxygen-radical production by the mechanism that involved endocytosis, whereas chemotactic peptide stimulated production by a mechanism that involved exocytosis of myeloperoxidase, the latter mechanism requiring an increase in intracellular free [Ca2+].     

Tissue phosphatase changes following triamcinolone associated with cleft palate in rats.

One theory of the development of cleft palate in rats involves the action of lysosomal enzymes secreted by epithelial cells at the time of fusion of the palatal shelves. To test this theory we studied the biochemistry of the palates of fetal rats daily between days 14 and 19 (from 3 days before to 3 days after palate closure). Triamcinolone was administered once im on gestation day 14 to Wistar rats; 0.5 mg/kg body weight produced approximately 50% cleft palates. Pooled control palatal tissue was compared with pooled experimental tissue; that from fetuses with clefts being pooled separately from those not affected. Acid phosphatase and beta-glucuronidase were assayed. Concentration vs. time curves for both enzymes were very similar. Prior to the time of palate closure both enzymes were present in low concentration. Between days 16 and 17, the normal time of closure, there was an abrupt increased in enzyme concentration, with experimental tissue showing a significant elevation over control tissue on days 17 and 18. Alkaline phosphatase was also present in small amounts before closure and significantly higher in control tissue on day 17. Protein was depressed in palates having clefts on day 17; thus the ratio of enzyme activities to protein synthesis was significantly elevated at a critical time. Unaffected experimental palates had a normal ratio. These results suggest imbalanced acid phosphatase, beta-glucuronidase, and alkaline phosphatase activity compared with protein synthesis at the time of palate closure following triamcinolone in rats.     

Effect of dexamethasone on polypeptides synthesised in polymorphonuclear leucocytes

The effect of a glucocorticoid on protein synthesis in human polymorphonuclear leucocytes (PMNLs) was investigated by two-dimensional gel electrophoresis. In the peripheral blood PMNLs of healthy laboratory personnel, the rate of incorporation of L-[35S]methionine into a least nine polypeptides was consistently influenced by dexamethasone in a dose-dependent manner, being increased in the case of seven polypeptides and decreased in the remainder.     

Ion-transporting ATPases and matrix mineralization in cultured osteoblastlike cells

Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca2+-ATPase; Ca2+,Mg2+-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na+,K+-ATPase activity was lower in treated than in untreated cells. HCO3--ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix.     

Coagulant properties of hybrid human/porcine factor VIII molecules.

Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.     

Alkaline phosphatase activity in human polymorphonuclear leukocytes

Synopsis Alkaline phosphatase has been localized ultracytochemically in PMN of man with normal and elevated levels of this enzyme. Contrary to guinea-pig PMN, no activity appears to be present in the specific granules. Instead, the plasma membrane and the membrane of the endocytic vacuoles show a strong staining. However, the demonstration of this activity depends on the preparatory procedure employed for PMN isolation. the use of dextran and Ficoll-Hypaque in the isolation procedure induces a marked increase in alkaline phosphatase staining of the PMN plasma membrane. Strongly increased activity at this site has been found in PMN from cancer patients. In most of them, additional staining has been observed in atypical vesicles and sometimes in the Golgi apparatus. These findings are discussed in the light of some previously reported controversial biochemical and cytochemical data on the distribution of alkaline phosphatase in human PMN.     

Alteration of human breast tumor cell membrane functions by chromosome-mediated gene transfer

BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 X 10^?5 and 1 X 10^?5, respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and survives on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 μM hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.     

Alkaline phosphatase in KB cells: influence of hyperosmolality and prednisolone on enzyme activity and thermostability

KB cells, derived from a human nasopharyngeal carcinoma, have high alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity. Specific activity is 150 times higher than in HeLa S3 cells. Alkaline phosphatase activity in KB cells exhibits heterogeneity consisting of a large heat-labile and a small heat-stable component. Enzyme activity is modulated by the osmolality of the culture medium. Increasing the osmolality by the addition of NaCl results in reduction of activity. A similar effect is noted when KB cells are grown with prednisolone (Δ′-hydrocortisone). The activities of acid phosphatase, β-galactosidase and N-acetyl-β-glucosaminidase are not affected by hyperosmolality and/or prednisolone. The reduction in alkaline phosphatase activity is accompanied by an increase in the proportion of the heat-stable and a decrease of the heat-labile enzyme components. The alterations in specific activity and thermostability are independent of the basal culture medium employed. Enzyme stability at 56 °C is inversely related to the buffer concentration, but the differences in stability between the alkaline phosphatases of control cultures and of KB cells grown in hyperosmolar medium or with prednisolone can be recognized with various buffering mixtures.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis

Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O₂ ^– and H₂O₂, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O₂ ^– and H₂O₂ by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H₂O₂ is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H₂O₂ degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H₂O₂ degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.     

Lysosomal hydrolases in neuronal, astroglial, and olidodendroglial enriched fractions of rabbit and beef brain.

Arylsulfatases A, B, and C, beta-galactosidase, and acid phosphatase were assayed in neuronal, astroglial, and oligodendroglial fractions isolated from adult rabbit and beef brains. The specific activities of all acid hydrolases were lower in beef cells compared to rabbit cells. The lysosomal enzymes of the rabbit neuronal fraction showed 10--25 time higher activities than the oligodendroglial fraction and 5-fold higher activities than the astroglial fraction. In beef brain, the specific activities of these enzymes were similar in oligodendroglia and astrocytes but 4--10 times lower than in neurons. The low activity of arylsulfatase A and beta-galactosidase in oligodendroglial cells may suggest that the low turnover of cerebroside and sulfatide in myelin may be regulated in part by the enzymes that catalyze their degradation.     

The interaction of Trichomonas vaginalis with epithelial cells, polymorphonuclear leucocytes and erythrocytes on vaginal smears: light microscopic observation

In this study, vaginal smears taken from 400 patients were examined cytologically using the Papanicolaou technique. Twenty of the 400 patients were detected as harbouring Trichomonas vaginalis. The interactions of T. vaginalis with epithelial cells, polymorphonuclear leucocytes (PMNLs) and erythrocytes were determined at light microscopic level. It was observed that T. vaginalis were juxtaposed to the epithelial cells and changed shape according to the contours of the epithelial cell revealing the cytopathic effect of trichomonads on epithelial cells. Trichomonads attached to PMNLs produced pseudopodia to phagocytose the cells. Occasionally an amoeboid shaped T. vaginalis organism was seen trailed by a row of PMNLs. This light microscopic study supports the production by trichomonads of a chemotactic factor for PMNLs. Phagocytosed erythrocytes were also seen in the cytoplasm of T. vaginalis, suggesting the need for the patient to be tested for anaemia.     

Culture and Characterization of Epithelial Cells from Bovine Choroid Plexus

Epithelial cells were isolated from choroid plexus, which plays a major role in cerebrospinal fluid production and regulation. Incubation of bovine choroid plexuses with pronase released cells which attached to plastic dishes with a plating efficiency of 5%. The cells were predominantly polygonal as judged by phase-contrast microscopy. These polygonal cells undergo limited cell division and survive for 1-2 weeks in culture before being overgrown by fibroblasts. The fibroblastic cells could be selectively removed from the cultures but the addition of 100 microgram/ml cis-hydroxyproline to the medium for several days. The specific activities of three membrane-bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were compared in selective cultures of polygonal cells and fibroblasts. Polygonal cells were found to have 4-5 times the gamma-glutamyl transpeptidase of fibroblasts, whereas fibroblasts have 2-3 times the alkaline phosphatase of polygonal cells. Leucine aminopeptidase levels in the two cultures were roughly equivalent. The polygonal cells rapidly lost gamma-glutamyl transpeptidase activity over a 4-day period in culture but acquired increased levels of leucine aminopeptidase. Alkaline phosphatase remained roughly constant. Under similar conditions fibroblasts showed a 3- to 4-fold increase in the specific activities of all three enzymes; these changes coincided with a substantial increase in cell density. Based on morphology, resistance to cis-hydroxyproline, absence of antihemophilic factor antigen, and enzymatic characteristics, we believe the polygonal cells to be of epithelial origin.     

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.     

Ion-transporting ATPases and matrix mineralization in cultured osteoblastlike cells

Summary Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca²⁺-ATPase; Ca²⁺,Mg²⁺-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na⁺,K⁺-ATPase activity was lower in treated than in untreated cells. HCO₃ ⁻-ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix. This work was supported by Grant NAG-2-108 from the National Aeronautics and Space Administration, Washington, D.C., and Grant 5 PO1 NS15767 from the National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD.     

Microanalysis of epithelial and mesenchymal acid hydrolase activities in the developing palate

Microdissection of lyophilized sections of fetal heads permits the analysis of relatively pure samples of epithelium and mesenchyme. These techniques were applied to a study of acid phosphatase and beta-glucuronidase activities in the developing palate in A/Jax mice. Acid phosphatase was found to be more concentrated in the palatal epithelium than the underlying mesenchyme. Conversely, beta-glucuronidase was more concentrated in the mesenchyme than the epithelium. A disparate developmental pattern of acid phosphatase and beta-glucuronidase activity was observed in the oral epithelium: acid phosphatase activity increased from intra-uterine Day 17 into neonatehood and beta-glucuronidase activity decreased towards term. Analysis of cortisone-induced palatal shelves showed increased activity of both acid phosphatase and beta-glucuronidase in the presumptive fusing epithelium.