Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (M?). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host M? was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 M? rather than to antigen-pulsed strain 13 M?. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 M? than F1 T cells that recognized antigen on strain 13 M?. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.     

Binding of sulfonamide and acetamide to the active-site Zn2+ in carbonic anhydrase: a theoretical study

Self-consistent field molecular orbital (SCF MO) calculations at both 4-31G and STO-3G levels have been used to examine the binding conformations of sulfonamide and acetamide compounds to the active site of carbonic anhydrase. The results are as follows: (1) sulfonamide binds to the Zn2+ ion in its deprotonated form through the sulfonamide nitrogen to the fourth coordination site of the metal ion; (2) acetamide as neutral species binds to the basic form of the enzyme through the carbonyl oxygen to the fifth coordination site of the metal ion; and (3) the acetamidate ion binds to the acid form of the enzyme through the amide nitrogen to form a tetracoordinated metal complex with three histidine ligands. Analysis of the effects of individual active-site residues on the binding conformations of these inhibitors suggests that metal alone favors bidentate coordination of sulfonamidate and acetamidate complexes and that electron donation from three histidine ligands to the metal ion determines the formation of a tetracoordinated metal complex, which is further stabilized by the presence of Thr 199, as it receives one hydrogen bond from the sulfonamide NH- or from the acetamide NH- and donates a backbone NH hydrogen bond to a sulfonamide oxygen. The calculated binding conformation of sulfonamide and the hydrogen-bonding interactions between sulfonamide and the enzyme are consistent with the X-ray diffraction study of the AMSulf-HCA II complex. However, no X-ray structures are available for amide-HCA II complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a single amino acid mutant of aspartate carbamoyltransferase at 2.5-A resolution: implications for the cooperative mechanism

One of the many interactions important for stabilizing the T state of aspartate carbamoyltransferase occurs between residues Tyr240 and Asp271 within one catalytic chain. The functional importance of this polar interaction was documented by site-directed mutagenesis in which the tyrosine was replaced by a phenylalanine [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. In the Tyr240----Phe mutant, the aspartate concentration required to achieve half-maximum velocity is reduced to 4.7 from 11.9 mM for the native enzyme. Here, we report an X-ray crystallographic study of the Tyr240----Phe enzyme at 2.5-A resolution. While employing crystallization conditions identical with those used to grow cytidine triphosphate ligated T-state crystals of the native enzyme, we obtain crystals of the mutant enzyme that are isomorphous to those of the native enzyme. Refinement of the mutant structure to an R factor of 0.219 (only eight solvent molecules included) and subsequent comparison to the native T-state structure indicate that the quaternary, tertiary, and secondary structures of the mutant are similar to those for the native T-state enzyme. However, the conformation of Phe240 in one of the two crystallographically independent catalytic chains contained in the asymmetric unit is significantly different from the conformation of Tyr240 in the native T-state enzyme and similar to the conformation of Tyr240 as determined from the R-state structure [Ke, H.-M., Lipscomb, W. N., Cho, Y. J., & Honzatko, R. B. (1988) J. Mol. Biol. (in press)], thereby indicating that the mutant has made a conformational change toward the R state, localized at the site of the mutation in one of the catalytic chains.     

Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms

The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry. The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155). We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively). The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain. The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations. The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees. No changes occur in secondary structure. Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit. However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme. Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains. The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism. The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding. On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o     

Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa

In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique of fluorescence recovery after photobleaching, we have demonstrated that a variety of fluorescent lipid analogues exhibit a nondiffusing fraction in the plasma membrane of the anterior region of the ram sperm head. The possible causes of this nondiffusing fraction were investigated. The nondiffusing lipid fraction is not the result of lipid oxidation during handling, and it is not released by extensive enzymatic digestion of the membrane surface proteins or the "bleeding" of the membrane by hypoosmotic shock. When lipid bilayers were prepared from protein-free lipid extracts of the plasma membranes of spermatozoa, most of the nondiffusing fraction was retained. These results suggest that the nondiffusing lipid fraction results from lipid factors such as lateral phase separations, which can cause such a nondiffusing fraction in model systems.     

4-Hydroxyphenylpyruvate dioxygenase is an iron-tyrosinate protein

A resonance Raman investigation into the blue chromophore of 4-hydroxyphenylpyruvate dioxygenase, a non-heme iron enzyme from Pseudomonas P. J. 874, reveals the presence of enhanced vibrations characteristic of tyrosinate coordination to the iron center. The excitation profiles for these features show that they are associated with the 595 nm absorption feature. EPR studies of this enzyme indicate the presence of a high-spin ferric center in a rhombic environment, as evidenced by a signal at g = 4.3 with the correct intensity for the measured iron content. This enzyme thus belongs to the emerging class of iron-tyrosinate proteins.     

Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH-benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe.     

Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies

The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5. The steady-state turnover rate (kcat) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9. Its change with pH conforms to a single protonation process with an apparent pKa of 7.3. The effect of pH on the apparent Michaelis constant (KMapp) of L-ornithine suggests that this diamino acid in its cationic form is not the substrate. Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (kcat/KMz) of the reaction is a bell-shaped curve characterized by pKa's of 6.2 and 9.1 and asymptotic slopes of +/- 1. Similar pKa's (6.3 and 9.3) are obtained for the pKi profile of zwitterionic L-norvaline, a competitive inhibitor. The pKi profile further indicates that the alpha-amino group of the inhibitor must be charged for binding. Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N(CH2)3CH(NH3+)COO-, binds the enzyme productively. The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack. Following such binding, the need for deprotonation of the delta-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible. Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction.     

Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate

The ability of aspartate transcarbamylase from Escherichia coli to catalyze carbamylation of amino acids other than the natural substrate, L-aspartate, was examined. Cysteine, cysteate, cysteinesulfinate, and 3-nitroalanine showed kcat values at pH 7 of 0.16, 0.58, 5.2, and 62 s-1, respectively, while kcat with aspartate was 320 s-1. In a parallel study, competitive inhibition constants of 3-nitropropionate, 3-mercaptopropionate, 3-sulfopropionate, and 3-sulfinopropionate were found to be high, about 0.1 M, compared with that of succinate, 0.56 mM. Although cysteinesulfinate had low activity as a substrate, the pH dependences of kcat and kcat/Km in H2O and D2O observed with the compound closely paralleled those of aspartate. The results of these studies suggest that substrate specificity and reactivity are achieved in part by a strong, highly specific interaction of one or more active site residues with the beta-carboxylate of L-aspartate. Unlike the sigmoidal kinetics found with aspartate, saturation of native aspartate transcarbamylase by cysteine sulfinate showed a lack of cooperativity, even under conditions of activation of the reaction by ATP and inhibition by CTP. The cysteinesulfinate reaction was increased 9-fold by the bisubstrate analog N-phosphonacetyl-L-aspartate. These results were interpreted in terms of an inability of cysteinesulfinate to cause the allosteric conformational change promoted by aspartate.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.