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PubMed1981

The identification of specific antigens in circulating immune complexes by an enzyme-linked immunosorbent assay: detection of bovine kappa-casein IgG complexes in human sera.

Circulating immune complexes have been implicated in the development of tissue injury in many chronic disease states, but in most instances the inciting antigens have not been identified. This report describes the development of a sensitive enzyme-linked immunosorbent assay which can be used to screen rapidly and simultaneously the immune complexes of numerous sera for the presence of a suspected antigen. The prototype antigen sought here was kappa-casein, a frequent participant in immune complex formation in IgA deficiency and in atopic diseases. The enzyme-antibody conjugate described here can detect as little as 0.11 ng/ml of kappa-casein; for immune complexes formed in vivo or in vitro, one can distinguish circulating complexes containing casein from complexes not containing this antigen. Using this reagent, a major antigen in the immune complexes of hypogamma-globulinemic patients treated with intravenous gamma-globulin was unexpectedly identified as kappa-casein. This method of specific immune complex analysis is suggested as a practical and effective approach to the elucidation of the antigenic constituents of immune complexes found in many diseases.

Absorption↗

PubMed1975

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12. I. Overall structure of the core complex.

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex which can be obtained with a unique polypeptide chain composition, has been investigated in solution with the X-ray small-angle technique. The molecular mass of the core complex of 3.78-10(6) daltons verifies the ratio of polypeptide chains of 16:16:16 of the three enzyme components, pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, present in the complex. In connection with the values obtained for the radius of gyration (156.5A), volume (1.07(7) A3) and amount of solvent associated with the complex (1.03 g/g) a loose packing of subunits in the complex has to be assumed. The maximum diameter of the core complex of 433 A, as determined from the correlation function, corroborates the large extension of the complex. The comparison of experimental and theoretical scattering curves reveals a relatively isometric overall shape of the core complex.

Escherichia coli↗

PubMed1985

Macrophage handling of soluble immune complexes: evaluation of mechanisms involved in the selective clearance of complexes from the circulation.

The binding and release of soluble guinea pig IgG2-containing DNPBSA-anti-DNP complexes and antigen-free, covalently-linked anti-DNP IgG2 oligomers of similar size, by guinea pig peritoneal macrophages, has been examined in the absence and presence of monomeric IgG2, of unrelated antibody specificity, or the monovalent hapten, DNP lysine. Complex binding was found to differ from the binding of the oligomers in that it was about twice as efficient and was essentially irreversible even in the presence of an inhibitor of ingestion, cytochalasin B. On the other hand, quantitative complex release could be achieved, in the presence of the ingestion inhibitor, by including 1.5mM DNP lysine in the medium. Complex handling by macrophages at 37 degrees C was also examined in the presence of monomeric IgG2, at its serum concn, and in the absence and presence of cytochalasin B. Inhibiting ingestion did not impair the capacity of the macrophages to take up complexes under these conditions. On the basis of these findings and previous reports that complexes bound to a receptor-bearing membrane undergo additional antibody-antigen bond formation [Dower et al., Biochemistry 20, 6326-6334 (1981a) and Leslie, Protides biol. Fluids 29, 431-434 (1982)] it is proposed that complex aggregation at the phagocyte surface may constitute the critical irreversible event required for the selective clearance of complexes in vivo. Other biological implications of receptor-mediated complex aggregation are also discussed.

Animals↗

PubMed1984

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Does a unifying model exist for the complexes from gram-negative bacteria?

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobacter vinelandii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15-19 U/mg and an overall yield of 40%. The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:1:0.5 for E1:E2:E3 respectively. Based on E2 this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 +/- 50 kDa, it is concluded that the complex as isolated from A. vinelandii is based on a tetramer of E2 chains. The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E2E3 subcomplexes with E1 resulted in maximum complex activity at an E1/E2 ratio of 1.5-1.6. Similar titrations of E1E2 subcomplexes with E3 resulted in maximum activity at an E3/E2 ratio of 0.45-0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three E1 dimers, one E2 tetramer and one E3 dimer. With excess of either E1 or E3 a decrease in activity is observed which indicates competition between these components for binding sites on E2. As shown before [Bosma, H.J., de Kok, A., Markwijk, B.W., and Veeger, C. (1984) Eur. J. Biochem. 140, 273-280], the isolated E2 component is composed of 32 peptide chains of 66 kDa each. Upon addition of E1 or E3, E2 dissociated into tetramers. Dissociation is complete upon the addition of four E1 dimers of four E3 dimers per E2 tetramer. Addition of E1 to saturated E2E3 subcomplex or E3 to saturated E1E2 subcomplex did not result in extra binding but rather in displacement of bound E3 or E1 respectively. It is therefore concluded that the binding sites of E1 and E3 to the E2 chains are either identical or so closely spaced that steric hindrance prevents simultaneous binding of both components. A model is presented based on the cubic structure of the isolated E2 component. In this model the 32 E2 peptide chains are arranged in tetramers in the corners of the cube. This model is discussed in connection with the existing model for the Escherichia coli complex.

Amino Acids↗

PubMed1988

Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex.

Commercially available kits (Mycobacterium avium Complex Rapid Diagnostic System and Mycobacterium tuberculosis Complex Rapid Diagnostic System; Gen-Probe, Inc., San Diego, Calif.) utilizing nucleic acid hybridization for the rapid identification of members of the M. avium-M. intracellulare complex and M. tuberculosis complex were evaluated by using 339 clinical and American Type Culture Collection (Rockville, Md.) isolates. The tests, which can be performed in approximately 2 h, use specific [125I]DNA probes complementary to the rRNAs of M. avium, M. intracellulare, and M. tuberculosis complex, the latter of which includes M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. The M. avium-M. intracellulare probes correctly identified 99 of 114 M. avium-M. intracellulare isolates, with 7 false-negatives and 8 false-positives, for a sensitivity of 93.4% and a specificity of 96.6%. After repeat testing, 110 of 114 were correctly identified, with 4 false-negatives and no false-positives, for a sensitivity of 96.5% and a specificity of 100%. The M. tuberculosis complex probe correctly identified 99 of 102 M. tuberculosis isolates, with 1 false-negative and 2 false-positives, for a sensitivity of 99% and a specificity of 99.2%. After repeat testing, 100 of 102 isolates were correctly identified, with no false-negatives and 2 false-positives, for a sensitivity of 100% and a specificity of 99.2%. Overall, there were 15 discrepant M. avium-M. intracellulare results, with 4 such results after repeat testing, and 3 discrepant M. tuberculosis complex results, with 2 such results after repeat testing. The Gen-Probe kits are highly sensitive and specific for use in identifying M. avium-M. intracellulare complex and M. tuberculosis complex isolates and will be useful in the clinical laboratory which can use the present radionuclide-containing kits cost effectively.

Cost-Benefit Analysis↗

PubMed1985

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b.

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.

Antibodies, Monoclonal↗

PubMed1975

Immune complexes in the spleen. The difference between competitive inhibition of immune complex trapping in spleen follicles and inhibition by paratyphoid vaccine.

Paratyphoid vaccine injected between 4 days and 3 hours before injection of labelled immune complexes (125-I-labelled BGG-anti-BGG), inhibits follicular trapping of these complexes in the mouse spleen. Inhibition is maximal when paratyphoid vaccine is given 1 day before, almost no label being found in the spleen follicles. No inhibition of follicular trapping of the complexes occurred when paratyphoid vaccine was injected simultaneously with the labelled immune complexes. Competitive inhibition was found when unlabelled immune complexes were given together with labelled immune complexes. Simultaneous injection of mice with paratyphoid vaccine and labelled immune complexes resulted in an additonal form of localization of the labelled immune complexes in the white pulp, heavily labelled clumps also appearing in the periarteriolar lymphocyte sheaths and follicles. The results are discussed in relation to the mechanism of immune complex trapping in spleen follicles.

Animals↗

PubMed1989

Capillary-localized low-affinity antibody-antigen complexes act as a focus for the deposition of high-affinity complexes.

The hypothesis that low-affinity antibody-antigen complexes localized in the glomerular capillary wall can act as a focus for the subsequent deposition of complexes containing high-affinity antibody was tested with three experimental systems: (1) Experimental zinc deficiency was used to modulate antibody affinity and to determine its effect on the development of glomerulonephritis. Low-affinity (LA) mice fed a zinc-containing diet (Zn+) produce low-affinity antibody and develop glomerulonephritis when injected daily with antigen. However, LA mice fed a zinc deficient diet (Zn-) produce high-affinity antibody and do not develop chronic glomerulonephritis. Furthermore, when LA mice fed on a Zn+ diet and given daily antigen injections for 25 days were then given a Zn- diet and 25 further daily antigen injections, they developed glomerulonephritis more severely than did control LA mice given Zn+ diet throughout the whole experiment; (2) Immune complex localization was induced in LA mice by daily injections of ovalbumin and then i.v. injection of preformed high affinity anti-DNP-DNP-HSA complexes. These localized in the glomerular capillary wall in ovalbumin-injected animals in contrast to their mesangial localization in controls; and (3) High-affinity mice (HA) were given injections of preformed high- or low-affinity anti-DNP-DNP-HSA complexes and then 50 daily injections of DNP-HSA. The localization of complexes in HA mice following daily antigen injection was markedly influenced by the immunochemical characteristics of the complexes initially injected. These results suggest that the capillary localization of small, low affinity antibody-containing antibody-antigen complexes acts as a focus for the subsequent localization of larger, high-affinity antibody-containing complexes.

Animals↗

PubMed1979

Soluble oligovalent antigen-antibody complexes. I. The effect of antigen valence and combining ratio on the composition of fluorescein-carrier anti-fluorescein complexes.

Soluble oligovalent antigen--antibody complexes were prepared and analysed by ultracentrifugation in order to study the effect of the combining ratio, antigen valence and concentration upon the size and molecular composition of the composition of the complexes. Fluorescein (F) conjugates of rabbit serum albumin (RSA) and thyroglobulin (RTg) were combined with high affinity rabbit anti-F antibodies to form soluble complexes. The effect of the combining ratio paralleled findings in precipitating systems in that the largest soluble complexes were found at equimolarity and mild molar antibody excess. Tetravalent antigen formed precipitates at combining ratios near equimolarity, whereas trivalent antigens failed to precipitate at similar concentrations. Complexes prepared near equimolarity were most sensitive to changes in concentration, higher concentrations leading to larger complexes. The Ab/Ag ratios of different-size complexes in the same preparation were remarkably similar. This ratio was dependent on the antibody--antigen combining ratio, was limited by antigen valence and was not affected by concentration differences. The data support the hypothesis that soluble complexes are formed in two steps. First, antigen and antibody combine to form subunits whose Ab/Ag ratio is determined by the combining ratio and antigen valence. These subunits then combine to form larger complexes in a manner analogous to polymerization.

Antibodies↗

PubMed1984

Influence of immune-complex size and antigen-antibody ratio on immune complex detection with monoclonal rheumatoid factor and C1q.

Stabilized aggregates of human IgG were prepared over a wide range of molecular weights. These fractions with increasing molecular weight were adjusted to the same molarity or the same protein concentration and were tested in the solid phase C1q and monoclonal rheumatoid factor immune complex assays. At constant molarity results were linearly correlated with the size of the aggregates. At constant protein concentration, results were also linearly correlated with the size of the aggregates in the lower molecular weight fractions, although the number of aggregates in the fractions decreased with increasing molecular weight. It is concluded that results in these assays can only be compared with respect to concentration if the immune complexes have identical sizes. Consequently, we studied the relative affinity of C1q and monoclonal rheumatoid factor for antigen/antibody immune complexes of different sizes or different antigen/antibody ratios, using model immune complexes composed of tetanus toxoid/anti-toxoid and streptolysine O/anti-streptolysine O. Compared to C1q, monoclonal rheumatoid factor was found to have higher affinity for smaller complexes, and for complexes with higher antigen/antibody ratio. Immune complex containing sera of 5 patients with connective tissue diseases, and one normal serum, were fractionated on a Sepharose 4B column. The binding patterns of the different fractions to solid phase C1q and monoclonal rheumatoid factor were quite variable and in only one of these sera monoclonal rheumatoid factor had the expected preference for small complexes and C1q for large complexes. Moreover a remarkably high binding to C1q and/or monoclonal rheumatoid factor of the monomeric IgG fraction was found in 4 out of 5 patient sera.

Antibodies, Monoclonal↗

PubMed1982

Spectroscopic studies on bleomycin-iron complexes with carbon monoxide, nitric oxide, isocyanide, azide, and cyanide and comparison with iron-porphyrin complexes.

The bleomycin-iron complexes with CO, NO, C2H5NC, OH-, N-3, CN-, and CH3NH2 were characterized by electronic, ESR, 1H-NMR, and Mössbauer spectroscopies and the findings were compared with the corresponding hemoprotein complexes. The 1H-NMR and Mössbauer features for the CO and C2H5NC adducts of the bleomycin-Fe(II) complex are consistent with an S = 0 ferrous assignment. The OH-, CH3NH2, and N-3 adducts of the bleomycin-Fe(III) complex show the ESR, 1H-NMR, and Mössbauer spectra typical of a low-spin Fe(III). The unique Mössbauer parameters of the bleomycin-Fe(II)-NO complex demonstrate mixing between the NO pi- and the Fe 3d-orbitals. The magnitude of the proton chemical shifts over +/- 50 ppm indicates a high-spin ferric type for the bleomycin-Fe(III)-CN complex. The Mössbauer parameters (delta EQ = 0.89 and delta = 0.48 mm/s) of the CN- adduct differ substantially from those of typical low-spin hemoprotein-cyanide complexes. Except for the CN- adduct, the Mössbauer and crystal field parameters of these bleomycin-iron complexes are similar to those of the corresponding hemoprotein complexes.

Azides↗

PubMed1985

Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands.

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1979

CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin.

The effects of pH upon infrared spectra [CO stretching frequency (vco) region] and visible spectra of the CO complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A are reported. The vco for leghemoglobin--CO complexes was 1947.5 cm-1 at neutral pH. At acid pH myoglobin-- and hemoglobin--CO complexes developed vco bands at 1966--1968 cm-1, whereas leghemoglobin--CO complexes developed vco bands at approximately 1957 cm-1. All pKapp co values determined by pH-dependent variation of vco fell in the range 4.0--4.6. The pKapp co values determined from visible spectra were consistent with vco-determined values except for that of myoglobin--CO (visible pKapp co = 5.8). The pKapp co values in the 4.0--4.6 range appear to be pK values of the distal histidines, while the visible pKapp co of myoglobin--CO appears to be the pK of a group other than the distal and proximal histidines. The data are consistent with a model in which protonation of the distal histidine permits protein-free heme FeCO geometry in leghemoglobin--CO complexes but not in myoglobin-- or hemoglobin--CO complexes. Thus the heme pockets of leghemoglobins appear to be more flexible than the heme pockets of myoglobin and hemoglobin. The effects of pH upon visible spectra of the O2 complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A also are reported. pKapp o2 values of approximately 5.5 (leghemoglobins) and 4.4 (hemoglobin) are probably the pK values of the distal histidines. Comparisons of pKapp o2 values with pKapp co values indicate a more flexible heme pocket in leghemoglobins than in hemoglobin. The O2 complex of leghemoglobin c2 differed significantly from the O2 complexes of leghemoglobins a and c1 in visible spectra and titration behavior. These differences might be associated with the small structural differences in the region between the E and F helixes of leghemoglobins.

Animals↗

PubMedDate not supplied

[Formation of nitrosyl complexes of nonheme iron (2.03 complexes) in animal tissues in vivo].

Formation or dinitrozyl non-haem iron complexes (2.03 complexes) in white rats' tissues in vivo has been studied. The formation of these complexes has been examined in the liver kidneys and small intestines when injecting p/o NaNO2 to the rats. Most of the complexes have been found in the liver. They have not been detected in the heart, spleen, muscle, cerebrum and marrow in vivo. 3-4-fold content of 2.03 complexes has been determined while introducing iron and NaNO2 in running water. This effect has not been found when introducing iron and NaNO2 simultaneously. The mechanism of 2.03 complex formation in the animal tissues in vivo has been proposed. According to this mechanism these complexes are formed in the tissues when Fe-NO from nitrozyl non-haem from complexes originating in blood passes into SH-group of protein in tissues. The formation of 2.03 complexes in vivo has been found in blood uniform elements.

Administration, Oral↗

PubMed1985

Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP.

It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells (Edery, I., Lee, K. A. W., and Sonenberg, N. (1984) Biochemistry 23, 2456-2462). Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of [alpha-32P]dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found that it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of [alpha-32P]dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.

Adenosine Triphosphate↗

PubMed1983

Localization of the membrane attack complex (MAC) in experimental immune complex glomerulonephritis.

The role of the membrane attack complex (MAC) as a mediator of renal tissue injury was evaluated in rats affected by bovine serum albumin (BSA)-induced immune complex glomerulonephritis. Immunofluorescence studies revealed concurrent deposits of IgG, BSA, C3, and the MAC along glomerular capillary walls, although the MAC manifested a more restricted distribution than that observed for immune complexes. Immunoelectron microscopic techniques were utilized to demonstrate immune complexes, C3, and the MAC within dense deposits in the subepithelial aspect of the basement membrane. Visceral epithelial foot processes were fused in areas overlying large dense deposits and exhibited intense staining for the MAC, lesser reactivity for C3 but IgG was absent from the foot process membranes. Smaller granular deposits of immune complexes, C3, and the MAC were observed in the subendothelial region of the lamina rara interna and the lamina densa. Immune complexes may activate the classical complement pathway causing diffuse injury to the glomerular basement membrane (GBM), allowing subepithelial accumulation of complexes. These observations implicate the MAC as a mediator of GBM and juxtaposed podocyte membrane injury, thereby contributing to disruption of the glomerular filtration barrier. IgG and C3 were demonstrated within tubulointerstitial regions on the surface of collagen fibers in close proximity to the tubular basement membrane (TBM) of proximal convoluted tubules. Within the TBM, C3 localization was prominent with diminished reactivity for the MAC, but IgG was not detectable. The demonstration of C3 and scant MAC deposits in the TBM of nonimmunized control rats without evidence of interstitial IgG and C3 deposits suggests that both nonimmune and immune processes play a role in the pathogenesis of extraglomerular lesions. Evidence derived from these morphologic studies indicates that the MAC is associated with injury to the GBM, foot process membranes of visceral epithelium, and the TBM. Further experiments designed to selectively enhance or inhibit the deposition of MAC and assess consequent renal dysfunction are required to substantiate hypotheses concerning the in vivo membranolytic potential of the MAC in experimental immune complex glomerulonephritis.

Animals↗

PubMed1989

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex.

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.

Animals↗

PubMed1987

Removal of glomerular deposits induced by either preformed immune complexes or by a chronic immune complex model in NZB/W mice.

The solubilization and removal of defined glomerular immune complex deposits by excess antigen was examined in NZB/W female mice. Glomerular deposits were induced by administering preformed immune complexes to young (2 to 4 mo) mice before they naturally acquired deposits from endogenous disease and to old (7 mo) mice with deposits from naturally acquired disease. The administration of excess antigen specifically removed deposits of preformed immune complexes in both groups. This was associated with a reduction in circulating large latticed complexes containing more than two antigen and two antibody molecules (greater than Ag2Ab2). Established deposits in old mice therefore did not interfere with removal of newly induced deposits of preformed immune complexes. Glomerular deposits were also induced in young mice by a chronic human serum albumin (HSA) immune complex model. The antigen in immune deposits induced by 2 wk of chronic antigen administration was solubilized and was removed within 48 hr of administering excess antigen. Circulating antibodies to the antigen were also reduced by excess antigen. Glomerular deposits of mouse immunoglobulin and complement were not significantly reduced by excess antigen but remained more intense than in mice of comparable age given preformed complexes. Thus deposits of other antigen antibody systems and possibly endogenous disease were induced by the chronic HSA immune complex model in NZB/W mice. However, defined antigen deposits within deposits containing multiple antigen antibody systems can clearly be removed by administering excess antigen.

Age Factors↗