Alkaline Phosphatase Secretion Research Articles

Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas.

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.

Cotranslational secretion of diphtheria toxin and alkaline phosphatase in vitro: involvement of membrane protein(s).

Crude messenger ribonucleic acid fractions isolated from Corynebacterium diphtheriae and Escherichia coli were translated in an E. coli in vitro protein-synthesizing system and yielded precursors of the secreted proteins diphtheria toxin and alkaline phosphatase, respectively. Addition of inverted E. coli inner membrane vesicles to the system during the initial stages of translation resulted in the intravesicular segregation of mature diphtheria toxin and alkaline phosphatase. Outer membrane vesicles or inner membrane vesicles whose cytoplasmic surfaces had been treated with pronase could not mediate transmembrane transfer of diphtheria toxin or alkaline phosphatase. However, inner membrane vesicles isolated from E. coli spheroplasts which had been treated with pronase and inner membrane vesicles complexed with ribosomes during pronase treatment were functional in transmembrane transfer. At temperatures below the phase transition of E. coli membranes, no intravesicular segregation of alkaline phosphatase or diphtheria toxin was observed. The precursor forms of each protein accumulated free from the vesicles. These results suggest that an inner membrane protein, exposed on the cytoplasmic surface, plays an integral role in secretion.

Enzyme secretion in [formula omitted] K12: Studies on alkaline phosphatase synthesis using an unsaturated fatty-acid auxotroph

The role of unsaturated fatty-acid starvation, and of the substitution of trans for cis fatty acids in the membrane phospholipid, on the secretion of alkaline phosphatase, has been investigated. A system in which alkaline phosphatase synthesis was initiated by a temperature shift has been used to obviate possible complications resulting from phosphate depletion. In contrast to earlier reports, we find (a) there is very little effect of unsaturated fatty-acid starvation on the synthesis of alkaline phosphatase; (b) the synthesis of both β-galactosidase and alkaline phosphatase synthesis was severely reduced below 27–30°C in cells grown on trans Δ 9 16:1 fatty-acid, compared to cells grown on the cis Δ 9 16:1 analogue. Thus no preferential effect on alkaline phosphatase synthesis was observed.

Mechanistic aspects of the transfer of nascent periplasmic proteins across the cytoplasmic membrane in Escherichia coli.

Spheroplasts, prepared according to an efficient procedure which is described, secrete a smaller protein fraction of the total cell content than that secreted by intact cells. However, more proteins are found associated to the cell envelopes derived from these spheroplasts than to those derived from intact cells.This result was confirmed when we measured, by specific immunoprecipitation, the amount of secreted and unsecreted alkaline phosphatase. Whereas at any time, at 30 °C, in intact cells 99.6% of the alkaline phosphatase produced is secreted and found in the periplasm [see A. Torriani (1968) J. Bacteriol. 96, 1200–1207], only 40% was secreted by our spheroplast preparation. We suggest that partial depletion of outer‐membrane‐bound protease (or proteases), involved in processing of precursors of secreted protein, is responsible for the significant defect of secretion of precursors which then remain membrane‐bound. Such an experimental protocol has been carried out at various temperatures and the secretion of alkaline phosphatase was assayed. This secretion was found to slightly vary above 22 °C whereas it dramatically decreased below 22 °C. Practically no phosphatase was secreted at 13 °C. However, the synthesis of alkaline phosphatase was not differentially altered at low temperatures compared to total proteins. The inactive presumed precursor of this enzyme which was then not secreted was found associated to the cell envelope. Similar results have been observed in intact cells above 22°C where only the energy of protein synthesis (ΔE= 79 kJ), which causes the elongation of nascent chains, is used for protein secretion; below 22 °C, in the temperature range of the disorder‐order transition of the lipid paraffin chains associated to membrane, a ΔE of 125 kJ is required for secretion of alkaline phosphatase. A ΔE of 146 kJ was found for this process to occur in the same temperature range in spheroplasts. When oleate was incorporated into the membrane of strain K 1059, an unsaturated fatty acid auxotroph, the same result as with wild‐type strains was observed, but a decrease in secretion at high temperatures was induced by this modification of fatty acid composition of the membrane. When elaidate instead of oleate was incorporated into K1059 membrane, the fall in alkaline phosphatase secretion occurred at 33 °C instead of 22 °C, which indicates that the physical state of the lipid phase is really involved in the secretion process. A hypothetical model for secretion of nascent periplasmic proteins, based on the data reported, is presented.

The Role of Polysaccharide in the Secretion of Protein by Micrococcus sodonensis

Abstract Cell suspensions of Micrococcus sodonensis (ATCC 11880) secrete 7 to 10 individual proteins including an alkaline phosphatase, a nuclease, and a protease. The appearance of enzyme activities in the extracellular medium is dependent on the co-secretion of at least one of several polysaccharides also elaborated by these cells. This conclusion is based on the following observations: (a) bacitracin, an inhibitor of polysaccharide biosynthesis, proportionately inhibits polysaccharide and protein secretion at concentrations which have no effect on intracellular protein synthesis; (b) a mutant, impaired in its ability to secrete proteins, is also impaired in its ability to secrete a glucosamine-rich polysaccharide; (c) alkaline phosphatase secretion by mutant protoplasts is enhanced in the presence of exogenously supplied polysaccharide; (d) inclusion of 10 mm glucosamine or 2-deoxyglucose (a variety of other monosaccharides were ineffective) in the medium suppresses the secretion of all three enzymes as well as the glucosamine-containing polysaccharide; and (e) restoration of alkaline phosphatase secretion in glucosamine-inhibited protoplasts is achieved by the addition of purified preparations of the glucosamine-containing polysaccharide. The inhibition of enzyme secretion by either glucosamine or 2-deoxyglucose can be completely prevented or reversed by addition of equimolar concentrations of glucose. The addition of albumin or casein to glucosamine-inhibited cells markedly enhances the rate of alkaline phosphatase secretion. These results suggest that during secretion polypeptide chains are vulnerable to proteolytic degradation and that co-secretion of polysaccharide protects the protein from proteolysis.

Hormonal Control of Intestinal Alkaline Phosphatase Secretion in the Dog

Intestinal alkaline phosphatase was released into the perfused small intestinal lumen of the dog upon intravenous injection of secretin and cholecystokinin. A direct hormonal effect on the intestinal mucosa is considered responsible for the enzyme release, since bile and pancreatic juice were excluded from the perfusion solution. A simultaneous increase in the alkaline phosphatase activity of both mucosal tissue and perfusion solution after hormonal stimulation suggests a direct potentiation of the secretory process rather than a simple washout phenomenon or increased enzyme extrusion through the mechanical effects of muscular contraction of the gut wall. Cholecystokinin exerted a much more pronounced effect on enzyme release than did secretin. Isoenzyme characterization studies, based on different susceptibilities to urea and L-phenylalanine inhibition and to heat inactivation, indicate similarities between canine and human intestinal alkaline phosphatase, and suggest that the mucosal and perfusion solution enzymes are identical.

Influence of Secretin and Cholecystokinin on Intestinal Alkaline Phosphatase Secretion

The intravenous administration of secretin and cholecystokinin in 38 human subjects was accompanied by a marked increase in the alkaline phosphatase activity present in duodenal juice. Cholecystectomized subjects exhibited similar responses to those without previous surgery. One subject, with a T-tube placed in the common bile duct, showed no significant increase in alkaline phosphatase concentration in hepatic bile after secretin and cholecystokinin injection, while the concentration of the enzyme in duodenal juice showed a marked increase after the injection of each hormone. Isoenzyme differentiation studies, based on different susceptibilities to inhibition by urea and L-phenylalanine, indicate an intestinal origin of the enzyme. It is proposed that secretin and cholecystokinin may exert a direct effect on the release of alkaline phosphatase from the brush border membrane of the intestinal epithelial cell, or, alternately, that the observed enzyme release may represent a secondary effect of an increase in the intraluminal content of bile salts and pancreatic enzymes.

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.

Cholinergic‐stimulated alkaline phosphatase secretion and phospholipid synthesis in guinea pig seminal vesicles

AbstractIncubation of slices of seminal vesicles of the guinea pig with cholinergic drugs led to an enhanced secretion of alkaline phosphatase. Incubation with carbamylcholine also stimulated the incorporation of P32 into the phospholipid fraction. Both cholinergic effects required a supply of energy since dinitrophenol was inhibitory. The stimulation of enzyme secretion and phospholipid synthesis by carbamylcholine was completely abolished by atropine. Omission of calcium ions from the incubation medium and pre‐treatment of the slices with ethylenediaminetetraacetic acid (EDTA) caused a marked reduction in alkaline phosphatase secretion induced by carbamylcholine but had no effect on incorporation of P32 into phospholipids. Adenergic agents such as epinephrine, norepinephrine and isoproterenol did not influence these two processes. Addition of cyclic AMP, dibutyryl cyclic AMP and a phosphodiesterase inhibitor was also ineffective. The incorporation of P32 into the various phospholipids of the seminal vesicle was examined. In the presence of carbamylcholine, there was a marked increase in the P32‐specific activities of phosphatidylinositol (nearly 6‐fold) and of phosphatidylserine (about 1.5‐fold). These observations indicate that the guinea pig seminal vesicle, a large hollow organ composed of a single layer of epithelium, is ideally suited for studies concerning the biochemistry of macromolecular secretion.

Characteristics of secretion of penicillinase, alkaline phosphatase, and nuclease by Bacillus species.

The distribution of alkaline phosphatase and nuclease activity between cells and medium was examined in one strain of Bacillus licheniformis and four strains of B. subtilis. Over 95% of both activities was found in the medium of the B. licheniformis culture, but in the B. subtilis cultures the amount of enzyme activity found in the medium varied with the strain and the enzyme considered. B. licheniformis 749 and its penicillinase magnoconstitutive mutant 749/C were grown in continuous culture with phosphorous as the growth-limiting factor, and the kinetics of penicillinase formation and secretion were examined. Nutrient arrest halted secretion (usually after a lag of about 30 min) in both the inducible and constitutive strains. Chloramphenicol did not eliminate secretion, but under certain circumstances reduced its rate. In the inducible strain treated with a low level of inducer, the rate of secretion was more affected by the rate of synthesis than by the level of cell-bound enzyme. During induction, the onset of accretion of cell-bound penicillinase and secretion of the exoenzyme were nearly simultaneous. It seems unlikely that a long-lived, membrane- or cell-bound intermediate is mandatory in the secretion of the three enzymes by Bacillus species. In the case of penicillinase secretion, there are at least two different phases. When penicillinase synthesis is proceeding rapidly, the rate of secretion is five to six times greater at equivalent concentrations of membrane-bound penicillinase than it is when penicillinase synthesis is reduced. The data require that any membrane-bound intermediate in the formation of exoenzyme be much shorter-lived in cells with a high rate of synthesis than in cells with a low rate. Either there are two separate routes for the secretion of penicillinase or the characteristics of the process vary substantially between the early stages and the declining phase of induction.