Effect of SP-B peptides on the uptake of liposomes by alveolar cells.

BACKGROUND: Exogenous surfactant has been accepted worldwide as a therapy of RDS in premature and term infants. Exogenous surfactant is usually derived from lung extracts containing phospholipids and the surfactant proteins SP-B and SP-C. Synthetic peptides of SP-B and SP-C are being tested with the aim to develop a completely synthetic surfactant preparation. Nevertheless, the effects of these peptides on the endogenous surfactant metabolism remain unknown. OBJECTIVES: The effect of synthetic SP-B peptides on uptake of surfactant-like liposomes was investigated in alveolar cells. Native SP-B and seven SP-B peptides were included: monomeric and dimeric SP-B(1-25) (Cys-11 --> Ala-11), SP-B(63-78)and Ala-SP-B(63-78) (Cys-71 --> Ala-71;Cys-77 --> Ala-77)and their serine mutants. METHODS: In vitro, alveolar macrophages (AM) and alveolar type II cells (ATII) were incubated with liposomes containing SP-B or one of its peptides. In vivo, rats received intratracheally various SP-B peptides (SP-B/lipid ratio 1:33 w/w) incorporated in fluorescent surfactant-like liposomes. One hour after instillation, AM and ATII were isolated and cell-associated fluorescence was determined using flow cytometry. Confocal laser microscopy was performed to ensure internalization of the liposomes. RESULTS: In vitro uptake by AM or ATII was not influenced by the SP-B peptides. In vivo, SP-B(1-25) and Ser-SP-B(1-25) increased the uptake by AM whereas dSP-B(1-25) decreased the uptake. Neither SP-B(1-25) nor dSP-B(1-25 )affected total uptake by ATII. The overall uptake by SP-B(63-78) variants was not changed. CONCLUSIONS: Surface-active synthetic SP-B peptides do not interfere with the normal uptake of surfactant by ATII.

Influence of phosphatidylglycerol on the uptake of liposomes by alveolar cells and on lung function.

The effect of phosphatidylglycerol on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages as well as the effect on endogenous surfactant function was studied in vivo. Healthy ventilated rats were intratracheally instilled with fluorescent labeled liposomes with different concentrations of phosphatidylglycerol. Lung function was determined by monitoring arterial oxygenation and, at the end of the experiment, by recording static pressure-volume curves. In addition, alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that, in the presence of cofactors (Ca(2+), Mg(2+)), phosphatidylglycerol stimulates the uptake by alveolar macrophages but hardly affects the uptake by alveolar type II cells. High concentrations of phosphatidylglycerol reduce the number of alveolar macrophages in the alveolar space and deteriorate lung function. On the other hand, the presence of cofactors protects the lung against the negative effects of phosphatidylglycerol on endogenous surfactant and alveolar macrophages. This study indicates that the phosphatidylglycerol concentration may play a fundamental role in the surfactant function and metabolism depending on the presence of so-called cofactors like calcium and magnesium; further study is needed to clarify the mechanisms involved.

Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro.

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.

In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages.

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70-75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.

Alveolar macrophages, surfactant lipids, and surfactant protein B regulate the induction of immune responses via the airways.

The influences of alveolar macrophages (AM) and pulmonary surfactant on the induction of immune responses via the airways were assessed. Mice were depleted of their AM by intratracheal instillation of multilamellar vesicles containing dichloromethylene-diphosphonate followed by intratracheal instillation of a T cell--dependent antigen, trinitrophenyl--keyhole limpet hemocyanin, in vesicles of various compositions. The primary immune response was determined in the spleen of these animals using an ELI-Spot assay. The secondary immune responses in the sera of the mice were assessed using enzyme-linked immunosorbent assays. An immune response was detected in animals depleted of their AM and intratracheally instilled with antigen in small unilamellar vesicles consisting of either phosphatidylcholine cholesterol or surfactant lipids. Incorporation of surfactant protein (SP)-B in the antigen vesicles enhanced the immune response, whereas SP-A or SP-C in the antigen vesicle did not have an effect. Strikingly, intratracheal instillation of SP-B containing antigen vesicles can induce an immunoglobulin M immune response in mice without depletion of AM. These results indicate that SP-B containing vesicles can enhance the induction of immune responses via the airways and further illustrate the important roles of both AM and pulmonary surfactant in the pulmonary immune system.

Functional immaturity of rat alveolar macrophages during postnatal development.

Alveolar macrophages (AM) are important in the regulation of immune responses in the lung, through their role as scavenger cells and through the production of many bioactive factors. Because in early infancy pulmonary infections are a recurrent problem, we studied the postnatal functional maturation of AM in a rat model. AM were isolated from rat lungs by bronchoalveolar lavage at several time intervals after birth and tested for their ability to ingest Escherichia coli in the presence of surfactant protein A (SP-A). Furthermore, their capacity to produce nitric oxide (NO) and interleukin-1 beta (IL-1 beta) after in vitro lipopolysaccharide (LPS) stimulation was analysed, as well as their capacity to downregulate proliferation of T cells from both mature and neonatal rats. SP-A-mediated phagocytosis of E. coli by AM was reduced in 14-day-old neonatal rats, as compared with mature rats (P < or = 0.05). Also the IL-1 beta production by rat AM after LPS stimulation was impaired at 14 days of age, as compared with IL-1 beta production by AM from mature rats (P < or = 0.05). In contrast, the LPS-induced NO production by rat AM as well as the capacity to inhibit T-cell proliferation were well developed at all ages tested. In conclusion, during postnatal development the rat AM is functionally immature, with respect to phagocytosis and secretion of inflammatory mediators. These differences may underly the enhanced susceptibility to pulmonary infections as found in human neonates.

Nitric oxide production by rat alveolar macrophages can be modulated in vitro by surfactant protein A.

Alveolar macrophage and type II cells are known to generate nitric oxide, which is a highly reactive molecule that plays a role in host defense against pathogens, as well as tissue damage associated with inflammation in the lung. Both types of cells are known to generate the nitric oxide by inducible nitric oxide synthase (iNOS). Surfactant-associated protein A (SP-A) from various sources (human alveolar proteinosis, rat and recombinant rat) was found to upregulate nitric oxide production by alveolar macrophages in a concentration- and time-dependent manner, whereas type II cells were unresponsive to SP-A. The increase in nitric oxide production was associated with elevation in the expression of iNOS. However, only 30-50% of the cells responded by expressing iNOS, as was observed by immunofluorescence staining. The stimulatory effect of SP-A was found to be 30-50% lower than the known nitric oxide agonists interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). However, addition of the cytokines interleukin-1 or granulocyte macrophage colony-stimulating factor elevated the levels of nitric oxide production to that of LPS and IFN-gamma. Special attention was given to exclude the possibility that contaminating LPS in the various SP-A species stimulated nitric oxide production by the macrophages. Our results indicate that SP-A is the agonist and not a contaminating LPS. The data presented in this report extend our knowledge regarding the nonsurfactant-related functions of SP-A.

Surfactant protein A, but not surfactant protein D, is an opsonin for influenza A virus phagocytosis by rat alveolar macrophages.

Surfactant protein A (SP-A) and surfactant protein D (SP-D) are collectins, and both proteins were shown to interact with influenza A virus and alveolar macrophages. However, it is not known whether SP-A and SP-D can serve as opsonins for the phagocytosis of influenza A virus by alveolar macrophages. In the present study, we investigated the opsonic activities of SP-A and SP-D for phagocytosis of fluorescein isothiocyanate (FITC)-labeled influenza A (H3N2) virus by rat alveolar macrophages using flow cytometry. SP-A enhanced the association of the virus with macrophages in a dose-dependent manner, reaching a maximum at an SP-A concentration of 60 microg/ml. An approximate threefold increase in association of influenza A virus with alveolar macrophages in the presence of SP-A over control incubations which contained no SP-A was observed. Half of the total cell-associated fluorescence could be quenched as demonstrated using the extracellular quenching dye trypan blue. These results indicate that SP-A mediates internalization of FITC-labeled influenza A (H3N2) virus by alveolar macrophages. Removal of the carbohydrate moiety of SP-A by N-glycosidase F treatment or cleavage of its sialic acid residues by neuraminidase abolished the enhancement of the phagocytosis of FITC-labeled influenza A virus by alveolar macrophages. Mannan, a mannose homopolysaccharide known to bind to the carbohydrate-binding domain of SP-A, did not affect the SP-A-mediated phagocytosis of FITC-labeled influenza by alveolar macrophages. In contrast, SP-D neither enhanced the association of FITC-labeled influenza A virus with alveolar macrophages nor affected the opsonic activity of SP-A for FITC-labeled influenza A (H3N2) virus at the SP-D concentrations tested. It is concluded that SP-A acts via its sialic acid residues as an opsonin in the phagocytosis of influenza A virus by alveolar macrophages.

Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.

To study the in vivo role of alveolar macrophages (AM) in gram-negative bacterial pneumonia in mice, AM were eliminated by the intratracheal (i.t.) administration of dichloromethylene diphosphonate encapsulated liposomes. Subsequently, the AM-depleted mice were infected i.t. with 100 CFU of Klebsiella pneumoniae, and the effects of AM depletion on survival, bacterial clearance, and neutrophil (polymorphonuclear leukocyte [PMN]) recruitment were assessed. It was shown that depletion of AM decreases survival dramatically, with 100% lethality at day 3 postinfection, versus 100% long-term survival in the control group. This increased mortality was accompanied by 20- to 27- and 3- to 10-fold increases in the number of K. pneumoniae CFU in lung and plasma, respectively, compared to those in nondepleted animals. This decreased bacterial clearance was not due to an impaired PMN recruitment; on the contrary, the K. pneumoniae-induced PMN recruitment in AM-depleted lungs was sevenfold greater 48 h postinfection than that in control infected lungs. Together with an increased PMN infiltration, 3- and 10-fold increases in lung homogenate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) levels, respectively, were measured. Neutralization of TNF-alpha or MIP-2, 2 h before infection, reduced the numbers of infiltrating PMN by 41.6 and 64.2%, respectively, indicating that these cytokines mediate PMN influx in infected lungs, rather then just being produced by the recruited PMN themselves. Our studies demonstrate, for the first time, the relative importance of the AM in the containment and clearance of bacteria in the setting of Klebsiella pneumonia.

Opsonic activities of surfactant proteins A and D in phagocytosis of gram-negative bacteria by alveolar macrophages.

Surfactant proteins A and D (SP-A, SP-D) can interact with lipopolysaccharide (LPS) and stimulate alveolar macrophages. The opsonic activities of SP-A and SP-D for bacteria with different types of LPS and alveolar macrophages were investigated. In flow cytometric studies with fluorescein-labeled rough (J5) and smooth (O111) Escherichia coli and rat alveolar macrophages, SP-A enhanced binding of J5 but not O111 bacteria to macrophages. Most importantly, SP-A enhanced ingestion of J5 bacteria by alveolar macrophages and subsequent bacterial killing. Immunoelectron microscopy demonstrated that J5 bacteria, the interface between the bacterium and the outer membrane of the alveolar macrophage, and ingested bacteria were heavily labeled with SP-A. In contrast, SP-D did not mediate phagocytosis. SP-A acted as an opsonin in the phagocytosis of rough LPS-containing bacteria by alveolar macrophages, emphasizing the possible role for SP-A in the alveolar defense system.

A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties.

Previous studies have shown that surfactant protein A (SP-A) derived from alveolar-proteinosis patients activates rat alveolar macrophages. However, it is not known if normal rat, dog and human SP-A can also stimulate alveolar macrophages. As alveolar-proteinosis SP-A has a slightly different structure from ordinary SP-A, it would be possible that the ascribed alveolar-macrophage-stimulating properties of SP-A are restricted to alveolar-proteinosis SP-A. To clarify this issue, we isolated SP-A from normal rat and dog pulmonary surfactants, using the same isolation technique commonly used for the isolation of alveolar-proteinosis SP-A, i.e. by butanol precipitation. In contrast with human alveolar-proteinosis SP-A, rat and dog SP-A obtained thus could not activate rat alveolar macrophages to produce oxygen radicals or enhance the phagocytosis of fluorescein isothiocyanate-labelled herpes simplex virus. However, rat, dog and normal human SP-A isolated by a novel method, involving extraction from pulmonary surfactant by using n-octyl beta-D-glucopyranoside and subsequent purification by cation-exchange chromatography, were able to elicit an oxidative burst in rat as well as normal human alveolar macrophages. In addition, dog and rat SP-A obtained thus stimulated the phagocytosis of herpes simplex virus by rat alveolar macrophages. These findings indicate that normal human, rat and dog SP-A have the same alveolar-macrophage-stimulating properties as human alveolar proteinosis SP-A. Dog and rat SP-A isolated by this novel method had the same Ca(2+)-dependent self-aggregation and lipid-aggregation properties as SP-A isolated by butanol precipitation. The new and milder isolation procedure yielded SP-A of high purity, as judged by SDS/PAGE and ELISA.

Interactions of surfactant protein A with influenza A viruses: binding and neutralization.

The interaction of pulmonary surfactant protein A (SP-A) with influenza A H1N1 and H3N2 viruses was investigated. SP-A is a sialated C type lectin with affinity for mannose residues. Flow cytometry showed that binding of fluorescein isothiocyanate (FITC)-labeled SP-A to H3N2 virus-infected cells was specific and time- and concentration-dependent. Oligosaccharides did not affect the binding of FITC-SP-A to the infected cells. Preincubation of H1N1 and H3N2 with SP-A resulted in a dose-dependent reduction of the infectivity of the viruses to cells. Removal of the carbohydrate moiety of SP-A by N-glycosidase F or cleavage of its sialic acid residues by neuraminidase prevented the interactions of SP-A with the viruses. It is concluded that SP-A binds to influenza A viruses via its sialic acid residues and, thereby, neutralizes the virus.

Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides.

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

Binding of surfactant protein A (SP-A) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of SP-A.

Pulmonary surfactant protein A (SP-A) has been shown to act as an opsonin in the phagocytosis of viruses by alveolar macrophages. To determine whether SP-A binds to viral proteins and which part of the SP-A molecule is involved in this interaction, binding studies were undertaken. SP-A was labeled with fluorescein isothiocyanate, and its binding to herpes simplex virus type 1-infected HEp-2 cells, as a model for virus-infected cells in general, was studied using flow cytometry. The binding of SP-A to virus-infected cells was saturable, reversible, and both time- and concentration-dependent, reaching a maximal level after 30 min at an SP-A concentration of 10 micrograms/ml. An approximately 4-fold increase in binding of SP-A to infected cells over control cells was observed. Yeast mannan, a mannose homopolysaccharide, did not influence the binding. However, heparin inhibited binding of SP-A in a concentration-dependent manner. In addition, heparin could also dissociate cell-bound SP-A, indicating that polyanionic oligosaccharides are involved in the binding of SP-A to virus-infected cells. Deglycosylated SP-A, obtained by digestion with N-glycosidase F, did not bind to infected cells. Heparin or deglycosylation of SP-A had no effect on the stimulation of alveolar macrophages by SP-A. It is concluded that the carbohydrate moiety of SP-A is involved in the recognition of viruses by SP-A and may play a role in the antiviral defenses of the lung.

Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages.

Rat surfactant protein D (SP-D) was shown to enhance the production of oxygen radicals by rat alveolar macrophages. This enhancement, which was determined by a lucigenin-dependent chemiluminescence assay, was maximal after 18 min at an SP-D concentration of 0.2 micrograms/ml. Surfactant lipids did not influence the stimulation of alveolar macrophages by SP-D, whereas the oxygen-radical production of these cells induced by surfactant protein A was inhibited by the lipids in a concentration-dependent manner.

Exposure of surfactant protein A to ozone in vitro and in vivo impairs its interactions with alveolar cells.

This study focused on the question of whether exposure of surfactant protein A (SP-A) to ozone affected properties of this protein that may be involved in regulating alveolar type II cell and alveolar macrophage functions. In vitro exposure of human or canine SP-A to ozone reduced the ability of this protein to inhibit phorbol-ester induced secretion of [3H]phosphatidylcholine by alveolar type II cells in culture. Ozone-exposed human SP-A showed a decreased ability to enhance phagocytosis of herpes simplex virus and to stimulate superoxide anion production by alveolar macrophages. Experiments with elastase showed that ozone-exposed canine SP-A was more susceptible to proteolysis. A conformational change of the protein could underlie this phenomenon. Surfactant isolated from ozone-exposed rats (0.4 ppm ozone for 12 h) was also less able to stimulate superoxide anion production by alveolar macrophages than surfactant from control rats, which suggested that SP-A in vivo was also susceptible to ozone. The results of this study suggest that SP-A-alveolar cell interactions can be inhibited by ozone exposure, which may contribute to the toxicity of ozone in the lungs.

Surfactant protein A is opsonin in phagocytosis of herpes simplex virus type 1 by rat alveolar macrophages.

In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothiocyanate-labeled herpes simplex virus type 1 (FITC-HSV-1) by rat alveolar macrophages and the effects of surfactant protein A (SP-A) on this process. The phagocytosis of FITC-HSV-1 by alveolar macrophages, which was studied as a model for virus phagocytosis in general, was strongly enhanced in the presence of SP-A. The SP-A-mediated phagocytosis was time and concentration dependent, reaching a maximal level after 15 min of incubation and at an SP-A concentration of 5 micrograms/ml. Using a fluorescence quenching technique, we could show that at least 65% of the viruses were indeed internalized by the macrophages. The addition of SP-A to the system was sufficient for the phagocytosis of FITC-HSV-1 by the alveolar macrophages, suggesting that SP-A acts as an opsonin. This hypothesis was further strengthened by the observation that F(ab')2 fragments of immunoglobulin G directed against SP-A could abolish FITC-HSV-1 phagocytosis by alveolar macrophages preincubated with SP-A. Comparing the opsonic capacity of serum and SP-A, SP-A proved to be twice as potent as serum in stimulating phagocytosis of FITC-HSV-1 by alveolar macrophages. Complement factor C1q, which is known to possess a similar collagen-like domain as SP-A, did not stimulate phagocytosis of FITC-HSV-1 by alveolar macrophages nor did it inhibit SP-A-mediated HSV-1 phagocytosis. This study demonstrates that SP-A may play an important role in the antiviral defenses of the lung.

Impairment of phagocytic functions of alveolar macrophages by hydrogen peroxide.

Hydrogen peroxide (H2O2) inhibited phagocytosis and superoxide anion production by rat alveolar macrophages. The inhibition was irreversible and concentration and exposure time dependent. The potential relationship between H2O2-induced biochemical perturbations and impaired alveolar macrophage phagocytic functions was investigated. Alveolar macrophage viability and Fc receptor binding capacity were not affected by H2O2. There was probably no correlation between a H2O2-induced rise in cytosolic [Ca2+] ([Ca2+]i) and the impairment of phagocytosis by alveolar macrophages, as was suggested by the following findings. First, the H2O2-induced rise in [Ca2+]i could be inhibited by chelation of extracellular Ca2+, whereas the H2O2-induced impairment of phagocytosis could not. Second, the H2O2-induced rise in [Ca2+]i was reversible, whereas the impairment of phagocytosis was not. And finally, a rise in [Ca2+]i by incubation of alveolar macrophages with the calcium ionophore A23187 did not affect phagocytosis. Various experiments suggested that ATP depletion may play an important role in the H2O2 toxicity for alveolar macrophages. Comparable concentrations of H2O2 caused an irreversible decrease both in cellular ATP and in phagocytosis and superoxide production by alveolar macrophages. In addition, time course of ATP depletion and induction of impaired alveolar macrophage function were similar. In view of the fact that the strong oxidant H2O2 may react with a large variety of biological substances, possible other toxic lesions may not be excluded as underlying mechanism for H2O2-induced inhibition of phagocytic functions of alveolar macrophages.

Control of rat-liver glutaminase by ammonia and pH.

Regulation by ammonia of phosphate-dependent glutaminase in isolated rat-liver mitochondria was studied at pH values near the cytosolic pH of 7.0. 1. Glutaminase activity, both in the absence and presence of bicarbonate, was completely dependent on the presence of ammonia. 2. Glutaminase activity, both in the absence and presence of bicarbonate, was strongly depressed by decreasing the pH of the incubation medium from 7.0 to 6.8 when the ammonia concentration was below 0.5 mM. 3. Bicarbonate stimulated glutaminase activity only in the presence of low concentrations of ammonia. 4. The data indicate that the reported inhibition of glutamine degradation in the perfused liver at low pH [e.g. Häussinger et al. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 995-1001] is due to a decreased affinity of glutaminase for ammonia.