Amount Of Alkaline Phosphatase Activity Research Articles

Aloe Vera-Derived Gel-Blended PHBV Nanofibrous Scaffold for Bone Tissue Engineering.

Today, composite scaffolds fabricated by natural and synthetic polymers have attracted a lot of attention among researchers in the field of tissue engineering, and given their combined properties that can play a very useful role in repairing damaged tissues. In the current study, aloe vera-derived gel-blended poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibrous scaffold was fabricated by electrospinning, and then, PHBV and PHBV gel fabricated scaffolds characterized by scanning electron microscope, protein adsorption, cell attachment, tensile and cell's viability tests. After that, osteogenic supportive property of the scaffolds was studied by culturing of human-induced pluripotent stem cells on the scaffolds under osteogenic medium and evaluating of the common bone-related markers. The results showed that biocompatibility of the PHBV nanofibrous scaffold significantly improved when combined with the aloe vera gel. In addition, higher amounts of alkaline phosphatase activity, mineralization, and bone-related gene and protein expression were detected in stem cells when grown on PHBV-gel scaffold in comparison with those stem cells grown on the PHBV and culture plate. Taken together, it can be concluded that aloe vera gel-blended PHBV scaffold has a great promising osteoinductive potential that can be used as a suitable bioimplant for bone tissue engineering applications to accelerate bone regeneration and also degraded completely along with tissue regeneration.

Osteoinduction of stem cells by collagen peptide-immobilized hydrolyzed poly(butylene succinate)/β-tricalcium phosphate scaffold for bone tissue engineering.

Bone substitute is a therapeutic approach to treat bone abnormalities. A scaffold serves mainly as osteoconductive elements. To facilitate a better biological performance, short collagen peptide was immobilized onto hydrolyzed poly(butylene succinate)/β-tricalcium phosphate (HPBSu/TCP) scaffolds. PBSu/TCP (80:20) scaffolds were fabricated by a supercritical CO2 technique, hydrolyzed with 0.6 M NaOH and conjugated with short collagen peptide tagged with or without red fluorescence. The surface morphology and porous structure of scaffolds were characterized by scanning electron microscopy and micro-computed tomography. Human mesenchymal stem cells were cultured onto the scaffolds and examined for osteogenic differentiation and biomineralization invitro by means of alkaline phosphatase activity, alizarin red staining, and reverse transcription-polymerase chain reaction. The PBSu/TCP and HPBSu/TCP scaffolds were successfully prepared. Scanning electron microscopy and micro-computed tomography results showed that the porosity was distributed throughout the scaffolds with the pore sizes in the range of 250-900 µm. Fluorescence microscopy demonstrated retention of tagged short collagen peptide on the scaffold. Mesenchymal stem cells adhered and grew well on the material. Under osteogenic induction, cells cultured on the short collagen peptide -immobilized scaffold significantly produced a greater amount of alkaline phosphatase activity and positive mineralization than those cultured on the control scaffold. The present results have shown that the short collagen peptide-immobilized HPBSu/TCP scaffold enhanced osteoinduction and biomineralization of stem cell-derived osteoblasts, possibly via stimulation of alkaline phosphatase activity. This suggests the potential use of osteogenic peptide-immobilized material in bone tissue engineering for correcting bone defects.

Fabrication of strongly attached hydroxyapatite coating on titanium by hydrothermal treatment of Ti-Zn-PO4 coated titanium in CaCl 2 solution.

Hydroxyapatite (HAp) coating was formed on zinc phosphate (Ti-Zn-PO4) coated Ti plates by hydrothermal treatment in CaCl2 solution at 200°C for 12h. Uniform surface coverage of the fabricated HAp coating was obtained by this method. SEM-EDX analysis of the adhesion test area showed that the presence of fractures only occurred in HAp crystals. On the other words cohesive fracture was seen in HAp coating layer formed on the Ti-Zn-PO4. The measured strength was around 42.3±17MPa. Rat bone marrow (RBM) mesenchymal stem cells were cultured and differentiation-induced on each sample (Ti plate, Ti-Zn-PO4 coated and HAp coated), and cell calcification properties were examined. Apparent differences in morphology and extension of the RBM cells were obtained, while the Ti-Zn-PO4 coated samples showed the highest cell number among all samples. After differentiation-induction, HAp coated samples showed the highest amount of alkaline phosphatase activity, and the highest level of cell calcification. Therefore, the hard tissue compatibility of Ti is improved by hydrothermally HAp coating of samples.

Alkaline phosphatase activity in whitefly salivary glands and saliva.

Alkaline phosphatase activity was histochemically localized in adult whiteflies (Bemisia tabaci B biotype, syn. B. argentifolii) with a chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate) and a fluorogenic substrate (ELF-97). The greatest amount of staining was in the basal regions of adult salivary glands with additional activity traced into the connecting salivary ducts. Other tissues that had alkaline phosphatase activity were the accessory salivary glands, the midgut, the portion of the ovariole surrounding the terminal oocyte, and the colleterial gland. Whitefly nymphs had activity in salivary ducts, whereas activity was not detected in two aphid species (Rhodobium porosum and Aphis gossypii). Whitefly diet (15% sucrose) was collected from whitefly feeding chambers and found to have alkaline phosphatase activity, indicating the enzyme was secreted in saliva. Further studies with salivary alkaline phosphatase collected from diet indicated that the enzyme had a pH optimum of 10.4 and was inhibited by 1 mM cysteine and to a lesser extent 1 mM histidine. Dithiothreitol, inorganic phosphate, and ethylenediaminetetraacetic acid (EDTA) also inhibited activity, whereas levamisole only partially inhibited salivary alkaline phosphatase. The enzyme was heat tolerant and retained approximately 50% activity after a 1-h treatment at 65 degrees C. The amount of alkaline phosphatase activity secreted by whiteflies increased under conditions that stimulate increased feeding. These observations indicate alkaline phosphatase may play a role during whitefly feeding.

Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy

Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, Fasta and Blast analysis revealed 100% identity to chick β 7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3′ untranslated region of β 7-tubulin. β 7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of β-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10 −6 mol/L) resulted in a higher rate of [ 3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of β-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

Use of Alkaline Phosphatase for the Analysis of Tannins in Grapes and Red Wines

Tannins are the most abundant class of phenolics in grape berries and are the predominant determinants of astringency in red wines. We have adapted a microtiter plate assay that was first described for persimmon tannin, so that it can be used for analysis of tannins in grapes and wines. Our modification incorporates a high salt wash step that is thought to remove non-specifically bound alkaline phosphatase enzyme that is used to detect tannin in the analysis. Application of the plate binding assay to fractions collected from a normal phase HPLC separation of seed tannins indicates that the assay only detects tannins having more than three flavan-3-ol subunits. The standard assay uses bovine serum albumin (BSA) as the protein bound to the microtiter plate, but we have found that casein or gelatin can be substituted for BSA in the assay. Results show that the microtiter plate assay can be used to monitor extraction of tannin from grape skins and seeds into a model wine solution. We have developed a new protein precipitation assay for grape and wine tannin that is based on the tannin9s ability to co-precipitate alkaline phosphatase and BSA from a mixture of the two proteins. The tannin-protein precipitate is pelleted by centrifugation and washed to remove residual unprecipitated alkaline phosphatase. The tannin-protein complex is then dissolved in a 1 <i>M</i> diethanolamine buffer pH 9.4 and the amount of alkaline phosphatase activity in the dissociated precipitate is determined by addition of <i>p</i>-nitrophenylphosphate substrate. The amount of alkaline phosphatase activity in the redissolved pellet was shown to be proportional to the amount of seed tannin used to form the precipitate. Because the solution assay is easy to perform and requires only a spectrophotometer, it should be suitable for use even in small winery laboratories.

Phosphate regulates the stability of skeletal alkaline phosphatase activity in human osteosarcoma (SaOS-2) cells without equivalent effects on the level of skeletal alkaline phosphatase immunoreactive protein.

Inorganic phosphate (P(i)) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells, but not by means of changes in transcription or release from the cell surface. The current studies were intended to determine whether (1) P(i) affected the inactivation of ALP activity in human osteosarcoma (SaOS-2) cells; and (2) P(i)-dependent changes in ALP-specific activity were associated with equal, concomitant changes in the level of ALP immunoreactive protein. The results of these studies revealed that P(i) increased the stability of skeletal ALP activity without equivalent effects on the level of ALP immunoreactive protein. An increase in P(i) (from 0 to 1.8 mmol/liter) caused a time-dependent increase in the amount of skeletal ALP activity in the SaOS-2 cells, without a parallel increase in the amount of skeletal ALP immunoreactive protein, and a decrease in P(i) (from 1.8 to 0 mmol/liter) caused a time-dependent decrease in the amount of ALP activity, without a significant decrease in the total cellular content of ALP immunoreactive protein. Together, these observations suggest that P(i) may alter the level of skeletal ALP activity in SaOS-2 cells by inhibiting a process of irreversible inactivation that does not effect equal, concomitant changes in the level of skeletal ALP immunoreactive protein.

Source of Alkaline Phosphatase Activity in Epithelial Lining Fluid of Normal and Injured F344 Rat Lungs

In pulmonary toxicity screening tests in which the epithelial lining fluid (ELF) is sampled by bronchoalveolar lavage (BAL), investigators have observed both increases and decreases in alkaline phosphatase (AP) activity in BAL fluid during an acute inflammatory response. Such alterations in AP activity are difficult to interpret because of uncertainties concerning the source of the AP. It could be coming from type II cells, or from influxing neutrophils or serum, along with transudation of proteins due to the increased permeability of the alveolar capillary barrier. To aid in determining the source of AP activity in ELF during an acute inflammatory response, AP in ELF from control F344 rats was compared to AP in ELF from endotoxin-treated or quartz-treated rats and to AP in serum, type II cells, neutrophils, and lungs of untreated rats. The isoelectric focusing pattern and the inhibition by amino acids, a chelating agent, and heat were used to characterize the AP from the different sources. The AP in ELF from control and from inflamed lungs had similar characteristics and closely resembled AP in type II cells, total lung, and neutrophils. It was concluded that type II cells, and not neutrophils, were the major source of AP in ELF for two reasons: (1) The amount of AP activity in neutrophils was approximately 40-fold less than in type II cells, and (2) earlier studies indicated an enhanced increase in AP activity in ELF in neutrophil-depleted versus nondepleted rats in response to α-quartz. Current evidence from this study and others suggests that AP in ELF is associated with secreted surfactant, indicating that alterations in ELF levels of AP may mean changes in the rate of surfactant secretion or turnover. Time course studies on the alterations in AP activity in ELF in relation to histopathological changes in alveolar epithelia and surfactant secretion are needed to further interpret the significance of AP activity in ELF.

The alkaline phosphatase in human plexus chorioideus

The content of alkaline phosphatase isozymes in various brain regions was determined by monoclonal immunocatalytic assays. The levels of the isozymes in human brain tissues were low compared with those in other human tissues, liver, kidney, bone, intestine and placenta. Plexus chorioideus in the brain, however, was found to express significant amounts of alkaline phosphatase activity. The purified isozyme from human plexus chorioideus demonstrated a single 70 kDa protein band on SDS-polyacrylamide gel which coincides with that of tissue-unspecific alkaline phosphatase from human liver. The isozyme expressed in the plexus was confirmed to be the tissue-unspecific alkaline phosphatase isozyme with regard to its reactivity with monoclonal antibodies specific for liver alkaline phosphatase, heat stability, and the inhibition by amino acids. This finding adds new dimensions to the functional role this isozyme may play.

Skeletal alkaline phosphatase specific activity is an index of the osteoblastic phenotype in subpopulations of the human osteosarcoma cell line SaOS-2

During continuous culture with serial passage, the human osteosarcoma cell line SaOS-2 showed a time-dependent decrease in skeletal alkaline phosphatase (ALP) activity. Because this was indicative of heterogeneity, subpopulations of SaOS-2 cells were isolated from replicate low-density cultures. The subpopulations were less heterogeneous and more stable (with respect to ALP) than the parent population. ALP specific activity in the subpopulations ranged from 0.05 to 2.3 U/mg protein, and cytochemical analyses indicated multiple steady-state levels of ALP activity per cell. The amount of ALP activity in SaOS-2 subpopulations was proportional to collagen production ([ 3H]proline incorporation into collagenase-digestible protein; r = .84, P < .005), and to parathyroid hormone (PTH)-linked synthesis of cyclic adenosine monophosphate (cAMP) ( r = .88, P < .01). From these data, we inferred that ALP activity in SaOS-2 cells can provide a useful index of the osteoblastic phenotype, and that ALP activity, collagen production, and PTH-linked adenylate cyclase were coordinately regulated in these osteoblast-like osteosarcoma cells (ie, selection of subpopulations for ALP activity coselected for collagen synthesis and PTH-linked synthesis of cAMP). Further comparative studies showed that micromolar fluoride concentrations stimulated cell proliferation ([ 3H]thymidine incorporation into DNA) in low-ALP SaOS-2 subpopulations, but not in high-ALP cells ( P < .001), and that this differential sensitivity to fluoride was associated with an inverse correlation between fluoride-sensitive acid phosphatase and ALP activities ( r = −.91, P < .001).

In vitro evidence that bone formation may be coupled to resorption by release of mitogen(s) from resorbing bone

Bone-derived proteins have been shown to stimulate the proliferation of bone-forming cells and to increase the rate of embryonic bone formation in vitro. The current studies were intended (1) to determine the tissue distribution of bone cell-active mitogen(s) in the embryonic chick, (2) to determine the cellular origin and the target cell specificity of the bone cell-active mitogen(s) in embryonic chick bone, (3) to determine whether the release of mitogenic activity from embryonic chick tibiae was proportional to bone resorption, and (4) to compare mitogenic activities prepared from different skeletal sources, with respect to M r, chemical stability, and mitogen activity kinetics. A bone cell-active mitogen(s) was identified in extracts of bone and cartilage but not in extracts of muscle, liver, intestine, or brain. (Mitogenic activity was determined as increased incorporation of 3[H]-thymidine into DNA in serum-free, calvarial cell cultures.) Together, the following three observations indicate an osteoblastic origin for the bone cell-active mitogen(s) in chick bone. First, the mitogen content of embryonic chick tibiae increased 4.5-fold, during eight days of serum-free in vitro growth ( P < .005). Second, conditioned medium (CM) from serum-free monolayer cultures of calvarial cells contained bone cell-active mitogen(s), but CM from parallel cultures of skin, liver, and intestinal cells did not. And, finally, the amount of bone cell-active mitogen(s) in calvarial cell CM was correlated with the amount of alkaline phosphatase (ALP) activity per cell, ie, an index of osteoblastic differentiation ( r = .92, P < .005). Treatment of calvarial cells with cycloheximide caused parallel decreases in protein synthesis and mitogen release ( r = .99, P < .01), indicating that the mitogen was not stored in calvarial cells. Mitogenic activities prepared from calvarial cell CM, tibial CM, and EDTA-extracts of tibiae increased 3[H]-thymidine incorporation in calvarial cell cultures, but not in parallel cultures of skin, liver or intestinal cells. Furthermore, the mitogenic response of calvarial cells to these activities was inversely proportional to ALP activity/cell ( r = .97, P < .001), suggesting a relative specificity for osteoprogenitor cells. In the presence of skeletal effectors (PTH and Cl 2MDP) the release of bone cell-active mitogen(s) from embryonic chick tibiae was proportional to bone resorption ( r = .915, P < .005) and inversely proportional to bone formation ( r = −.80, P < .05). When cycloheximide was added to this system we found that mitogen release was still correlated with resorption ( r = .996, P < .001), but not with protein synthesis ( r = .129), suggesting that the mitogen in CM did not reflect newly synthesized protein. Bone cell-active mitogen(s), prepared from calvarial cell CM, tibial CM, and EDTA-extracts of chick bone, could not be distinguished from each other on the basis of M r, chemical stability, or mitogen activity kinetics. Together, these data indicate that a bone cell-active mitogen(s) is produced by osteoblasts, deposited in bone, released in proportion to resorption, and relatively specific for osteoblast progenitor cells, and these observations are consistent with the hypothesis that osteoblasts may produce a delayed paracrine effector of bone volume. Since we also observed that the amount of ALP activity in calvarial cell cultures was directly proportional to mitogen production, but inversely proportional to mitogen response, these data further indicate that this mitogenic activity may be an acute paracrine effector of calvarial cell proliferation in vitro.

Characterization of a rapidly responding animal model for fluoride-stimulated bone formation

Sodium fluoride (NaF) is the single most effective agent for increasing bone volume in the osteoporotic skeleton. However, the mechanism of fluoride-stimulated bone formation is not known, and investigation has been hampered by the lack of a suitable animal model. Young chicks show a rapid skeletal response to NaF that resembles the human skeletal response. This occurs at serum fluoride concentrations comparable to those obtained in humans. Fourteen-day-old chicks treated with NaF (4.2 mM NaF in the drinking water) for 2 weeks showed increases in bone-forming surface in the tibial metaphysis (130% of untreated controls, P < 0.002), with no change in the number of osteoblasts per length of forming surface (104% of control). The NaF dose dependence of the change in bone-forming surface was biphasic, being optimal at 23 μM fluoride. Linear correlations were observed between dietary NaF and serum fluoride ( r = 0.996, P < 0.001), and serum fluoride and bone fluoride concentrations ( r = 0.98, P < 0.001). Correlations were also observed between the amount of alkaline phosphatase activity in the tibia and the serum fluoride concentration ( r = 0.88, P < 0.03), the serum fluoride concentration and the tibial ash weight ( r = 0.93, P < 0.01), and the bone fluoride concentration and tibial ash weight ( r = 0.95, P < 0.01). Preliminary studies of the time dependence of the skeletal fluoride response in young chicks revealed no difference between 2 weeks and 4 weeks of treatment (bone-forming surface increased to 124% and 139% of controls in separate studies, P < 0.01 for each). The rapidly growing chick skeleton differs in some respects from the remodeling human skeleton with osteoporosis. Nevertheless, chicks, like humans, respond to NaF treatment with an increase in bone-forming surface, suggesting that the young chick may be a useful model to study the mechanisms of fluoride-stimulated bone formation in vivo.

Effects of 1,1-bis ( p-chlorophenyl)-2,2,2-trichloroethane (DDT) on gonadal development in the chick embryo: A histological and histochemical study

Fertile white Leghorn chicken eggs were exposed via intravitelline injections to dosages of 5.0, 10.0, or 20 mg 1,1-bis( p-chlorophenyl)-2,2,2-trichloroethane (DDT) in olive oil prior to incubation. Control embryos received only the olive oil vehicle. Eggs were placed in a forced-draft incubator for either 5 or 12 days. Embryos were removed and their gonadal areas prepared for histological or histochemical evaluation. Histological examination of DDT-exposed 5-day embryos revealed no significant differences in the number of primordial germ cells aggregating in the gonadal area and in the localization of acid and alkaline phosphatase activity. Embryos exposed to DDT for 12 days revealed significant alterations in both ovaries and testes. The testes of DDT-exposed embryos consisted of mostly stroma with fewer seminiferous cords than controls while ovaries of exposed embryos contained a larger number of distended medullary cords as well as a difference in the distribution of these cords when compared to controls. There was an increased alkaline phosphatase activity in the stromal cells of female gonads. Increased amounts of alkaline phosphatase activity found in the stroma at 12 days might be due to a DDT-induced stimulation of these cells to differentiate more rapidly. Acid phosphatase activity was found in the secondary sex cords of control 12-day ovaries, but was much reduced or absent in those of pesticide-exposed embryos. These results indicate that a single dosage of DDT administered to a chick embryo prior to incubation does not affect early stages of gonadal development but that effects on both ovaries and testes occur 12 days following exposure.

Evidence for the retinoid control of urothelial alkaline phosphatase

The amount of alkaline phosphatase activity per μg of DNA in the urothelium (transitional epithelium) of the rat urinary bladder, organ-cultured in chemically-defined serum-free medium, decreased greater than 70% during a 13 day culture period. This decrease in enzyme activity corresponded inversely with the increase in cell number in the urothelium indicating that enzyme synthesis did not accompany growth. Alkaline phosphatase activity was increased back to values approaching normal enzyme levels during a 3 day culture period by the addition of 10 μM retinoic acid. Retinol also increased enzyme activity but it was only half as effective as retinoic acid. A significant increase in enzyme activity was initiated by 1 μM retinoic acid, however the most effective concentration was at 10 μM.

Osteosarcoma Cells in Tissue Culture

Although elevated alkaline phosphatase levels in osteosarcoma have been shown to be related to prognosis, the functional significance is unclear. Human osteosarcoma cells in tissue culture retain detectable amounts of alkaline phosphatase activity. In this study the specific activity of that enzyme was compared in 13 osteosarcoma tissue culture lines and 13 normal skin fibroblast lines derived from the same patients. Osteosarcoma cells had significantly higher baseline alkaline phosphatase levels and could be stimulated with hydrocortisone to produce more enzymatic activity than the fibroblast lines. Activity was localized ultracytochemically to the cell membrane and to many small intracellular vesicles in stimulated osteosarcoma cells. These observations aid in the differentiation of osteosarcoma and fibroblast lines in tissue culture and suggest an association between elevated alkaline phosphatase levels and metabolic abnormalities in patients with osteosarcoma.

Ultrastructural localization of alkaline phosphatase and ATP-ase in cyst stages of Sarcocystis tenella (Sporozoa, Coccidia) parasitic in the esophagus of sheep (author's transl)

The activity of adenosine triphosphatase and alkaline phosphatase was investigated at the fine structural level in the cyst stages of Sarcocystis tenella parasitic in the esophagus of sheep. Alkaline phosphatase reaction was observed along the outer membrane of the parasite's pellicle. The enzymatic activity was much higher on the surface of metrocytes than that of zoites, which proved to be infectious. No reaction was noted in the interior of the parasites. However, a significant amount of alkaline phosphatase activity occurred along the inner surface of the 25 nm thick primary layer of the cyst wall. No evidence of the reaction of this enzyme was seen in the secondary cyst wall, which consisted of degenerated host cells. ATP-ase activity was found in a considerable degree along the primary cyst wall (=directly limiting the cyst's interior), whereas the ground-substance of the cyst, surrounding the parasites, is free of deposits. In the parasites ATP-ase was localized in the endoplasmic reticulum, in the perinuclear space, and between the two inner membranes of the three-layered pellicle. Only rarely a slight reaction was seen in the mitochondria of the metrocytes, which are the reproductive cells. The other organelles typical for S. tenella were free of ATP-ase. The results indicate that the enzymes studied participate in the growing process of the cysts, in which finally the infectious zoites remain in a more or less inactive state. The localizations of the enzymes corresponded with the results known from metazoa.

Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis: II. FACTORS AFFECTING SECRETION

The secretion of alkaline phosphatase by Micrococcus sodonensis has been studied by observing the production and accumulation of extracellular enzyme by log phase cells which have been resuspended in fresh growth medium. The accumulation of extracellular alkaline phosphatase activity is the result of a selective permeation process and is totally dependent upon the presence of divalent cation. Although calcium is an actual component of the enzyme and is required for both catalytic activity and enzyme stability, magnesium appears to be the divalent cation required for the actual synthesis or release (or both) of alkaline phosphatase from the cell. Studies with various inhibitors of protein synthesis suggest that the bacterial cell may possess at least two distinct sites of protein synthesis, one site functioning to provide intracellular and cell-associated proteins and another for the synthesis of extracellular proteins. The over-all process of enzyme secretion is markedly temperature-dependent and is characterized by an activation energy of 55 kcal per mole. This observation suggests that the rate-limiting step in alkaline phosphatase secretion is probably not an enzyme-catalyzed event. A relatively small but significant amount of alkaline phosphatase activity exists in a cell-bound form and can be quantitatively released from cells by treatment with lysozyme. Although lysozyme-sensitive cell wall components are involved in the binding of alkaline phosphatase to the cell, the cell wall is not an obligatory component of the secretion process since lysozyme-prepared, sucrose-stabilized protoplasts are capable of extracellular enzyme production.