Subunit Of Component Research Articles

High Doses of Intravenous Immunoglobulin Do Not Affect the Recognition Phase of the Classical Complement Pathway

We have recently found that intravenous immunoglobulin (IVIg) prevents deposition of C3 and C4 fragments onto antibody sensitized erythrocytes. To find out if such an effect results from the blockade of the recognition phase of the classical complement cascade, we investigated the ability of human serum containing high concentrations of IVIg to deposit the recognition subunit of the first complement component (C1q) onto targets. Normal human serum supplemented in vitro with IVIg did not demonstrate reduced C1q binding to targets as determined by radiolabeled antihuman C1q antibody uptake. Similarly, methylamine-treated normal human serum to which IVIg was added was equally effective in terms of C1q binding as the same serum without IVIg. At increasing doses of sensitizing antibody, C1q uptake decreased proportionally; however, at all antibody dilution points C1q uptake was not significantly different in the serum with IVIg in comparison with normal serum. Serum from a patient treated with IVIg did not differ in its capacity to deposit C1q from the same patient's serum before therapy. Our data suggest that IVIg does not interfere with the recognition step of classical complement pathway. This is a US government work. There are no restrictions on its use.

High doses of intravenous immunoglobulin do not affect the recognition phase of the classical complement pathway

We have recently found that intravenous immunoglobulin (IVIg) prevents deposition of C3 and C4 fragments onto antibody sensitized erythrocytes. To find out if such an effect results from the blockade of the recognition phase of the classical complement cascade, we investigated the ability of human serum containing high concentrations of IVIg to deposit the recognition subunit of the first complement component (C1q) onto targets. Normal human serum supplemented in vitro with IVIg did not demonstrate reduced C1q binding to targets as determined by radiolabeled antihuman C1q antibody uptake. Similarly, methylamine-treated normal human serum to which IVIg was added was equally effective in terms of C1q binding as the same serum without IVIg. At increasing doses of sensitizing antibody, C1q uptake decreased proportionally; however, at all antibody dilution points C1q uptake was not significantly different in the serum with IVIg in comparison with normal serum. Serum from a patient treated with IVIg did not differ in its capacity to deposit C1q from the same patient's serum before therapy. Our data suggest that IVIg does not interfere with the recognition step of classical complement pathway. This is a US government work. There are no restrictions on its use.

Characterization of cDNA clones for the beta subunit of pyruvate dehydrogenase from Ascaris suum

The pyruvate dehydrogenase complex occupies a unique position in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. This paper describes cDNA clones for the β subunit of the pyruvate dehydrogenase component of this complex. A cDNA library has been constructed in λgt11 from poly(A) + RNA isolated from adult ascarid body wall muscle. The library was screened with antiserum prepared against the β subunit of pyruvate dehydrogenase. Full-length clones of 1.2 kb have been characterized. The first 15 amino acids determined from the purified protein match exactly those predicted from the cDNA sequence. The deduced protein sequence contains a 26-amino-acid presequence that has characteristics of mitochondrial targeting sequences. The mature protein is predicted to contain 334 amino acids and is 62% identical to the predicted sequence of the corresponding human subunit [10]. Full-length in vitro transcripts have been translated in vitro to yield a 39-kDa polypeptide consistent with the open reading frame present in the cDNA sequence. The 90 nucleotides at the 3′ end of the cDNA sequence have the potential to form a cruciform structure that may play a role in the synthesis of the enzyme.

Glucose regulates leucine-induced insulin release and the expression of the branched chain ketoacid dehydrogenase E1 alpha subunit gene in pancreatic islets.

Much evidence has accumulated to support the idea that leucine can stimulate insulin release by allosterically activating glutamate dehydrogenase thus enhancing glutamate metabolism. It is less clear how the metabolism of leucine itself contributes to the signal for insulin release. We recently found that culturing pancreatic islets for 1 day at low glucose (1 mM) suppressed glucose-induced insulin release, but preserved leucine-induced insulin release. When islets were cultured at high glucose (20 mM), glucose-induced insulin release was preserved, but leucine-induced insulin release was suppressed (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol., 259, E548-E554). The suppression of leucine-induced insulin release can be explained by glucose's suppression of the synthesis of the enzyme that catalyzes the first committed step of leucine metabolism, branched chain ketoacid dehydrogenase complex (BCKDH). High glucose suppressed the enzyme activity of the E1 component of the BCKDH complex, as well as the total activity of the BCKDH complex, to usually negligible levels in islets and decreased by an average of 90% the mRNA which encodes E1 alpha, the catalytic subunit of the E1 component of BCKDH, in islets and rat insulinoma cells. Time course studies showed that about 24 h in culture was required to maximally induce or suppress the expression of BCKDH E1 alpha. Culture at high glutamine with or without leucine mimicked to a lesser and more variable degree the effects of high glucose on leucine-induced insulin release and BCKDH E1 alpha mRNA. Leucine-plus-glutamine-induced insulin release was present after culture of islets with glucose and with or without any other secretagogue. Also, glutamate dehydrogenase transcripts and enzyme activity were not significantly altered by varying the concentration of glucose in the culture medium. Thus, leucine's insulinotropism via activation of glutamate dehydrogenase is constitutive. Preproinsulin mRNA levels were markedly increased at high glucose and glyceraldehyde phosphate dehydrogenase transcripts were either unaffected or slightly increased by glucose. Glutamine did not significantly effect the expression of genes other than BCKDH E1 alpha, and leucine had little or no effect on the expression of any of the four genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase

1.1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin.2.2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity.3.3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5′-0-(2-thiodiphosphate) or guanyl-5′-yl imidodiphosphate.4.4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold.5.5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc mouse lymphoma membranes.6.6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the a subunit of the stimulatory guanine nucleotide-binding regulatory component (αGs).7.7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of αGs.8.8. Endogenous ADP-ribosylation of gaGs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP.9.9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.

Functional regulation of brain pyruvate dehydrogenase: Postnatal development, anesthesia and food-deprivation

Abstract The activity of brain pyruvate dehydrogenase complex (PDHC), is regulated by reversible phosphorylation of the alpha subunit of the E1 component (pyruvate dehydrogenase, EC 1.2.4.1) of PDHC. Using an in vitro back-titration assay, we have evaluated the postnatal development of E1α phosphorylation, as well as the effects of acute pentobarbital administration and food-deprivation on cerebral cortical E1α phosphorylation in synaptosomal and free mitochondrial compartments of the albino rat. Between birth and postnatal day 25, the back-titration phosphorylation increased ca 4-fold, with the largest increase occurring between days 15 and 20. The phosphorylation of E1α in the synaptosomal, but not free mitochondrial fraction, was decreased during pentobarbital anesthesia. Following 72 h of food-deprivation, E1α phosphorylation was decreased in both subcellular fractions. The postnatal increase in E1α back-titration phosphorylation is consistent with and similar in magnitude to previously reported increases in the specific enzymatic activity of PDHC. These results also highlight the potential importance of localized subcellular alterations in mitochondrial metabolism and further validate the back-titration phosphorylation of E1α as a valuable tool for the study of central nervous system PDHC metabolism.

Elusive Proximal Signals of β-Cells for Insulin Secretion

The beta-cell is unique because its major agonists, i.e., insulin secretagogues, undergo metabolism instead of interacting with a receptor. This perspectives presents the hypothesis that the first part of a metabolic signal of a secretagogue is specific to the secretagogue and the beta-cell and can be envisioned as proximal. The second part, which occurs after transduction to more universal signaling mechanisms, is viewed as distal. Distal signaling and exocytosis in the beta-cell operate the same as in other cells. Aerobic glycolysis is required for glucose-induced insulin release. Because glyceraldehyde, which enters metabolism at the triose phosphates in the glycolytic pathway, is a potent insulin secretagogue but pyruvate, which is metabolized in the mitochondrion, is not an insulin secretagogue, the proximal signal for glucose-induced insulin release originates with an interaction between the central part of the glycolytic pathway and mitochondrial metabolism. The proximal message in leucine-induced insulin release originates with leucine allosterically activating glutamate dehydrogenase, which activates endogenous glutamate metabolism, and by the metabolism of leucine itself. The methyl ester of succinate is a potent experimental insulin secretagogue. It is puzzling why the glucose signal requires the interplay of glycolysis and mitochondrial metabolism, whereas the signals from leucine and succinate originate entirely from within the mitochondrion. Leucine-induced insulin release is suppressed and glucose-induced insulin release is activated in islets cultured at a high concentration of glucose. Conversely, leucine-induced insulin release is activated and glucose-induced insulin release is suppressed in islets cultured at low glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor β enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast‐like cells

The effects of transforming growth factor beta (TGF beta) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Purified TGF beta incubated with UMR-106 cells for 48 hr produced a concentration-dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGF beta. TGF beta also enhanced receptor-mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptor-mediated enzyme activation by cholera toxin and forskolin. In cells in which PTH-stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGF beta was reduced by 33%. However, TGF beta neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP-ribosylation of a 41,000-Da protein, presumably the alpha subunit of the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase, in cholate-extracted UMR-106 cell membranes. TGF beta also had no effect on the levels of alpha or beta subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (less than or equal to 30 min) exposure of cells to TGF beta during early culture was sufficient to increase PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. TGF beta enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGF beta enhances PTH response in osteoblast-like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGF beta may involve Gi, although in a manner unrelated to either pertussis toxin-catalyzed ADP-ribosylation of the alpha subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGF beta on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGF beta may regulate osteoblast responses to systemic hormones.

Identification of the Heterozygotes for Deficiency of the β-Subunit of the Eighth Component of Complement by Reduced Levels of C8β and Increased Amounts of Free C8α-γ

Sera from obligate heterozygotes for deficiency of the C8 beta subunit of the eighth component of human complement (C8) were analyzed for the molecular composition of C8. The C8 alpha-gamma and C8 beta subunits were separated by SDS-PAGE, visualized by immunoblotting, and the resulting bands were quantitated by laser densitometry. The laser densitometric absorption data were set to 100 arbitrary units (AU) for both subunits of pooled normal human sera. The AU values of individual normal sera ranged from 45 to 150 AU for C8 alpha-gamma (median 99 AU) and from 45 to 140 AU for C8 beta (median 101), whereas the C8 alpha-gamma/C8 beta-ratio varied from 0.7 to 1.4. Sera from C8 beta-deficient heterozygotes differed, as expected, from the normal sera for the markedly reduced levels of C8 beta (20 to 90 AU, median 55 AU) and for the higher C8 alpha-gamma/C8 beta-ratio (1.3 to 3.5). High voltage agarose gel electrophoresis was used to separate free and C8 beta-bound C8 alpha-gamma. The migration of free and C8 beta-bound C8 alpha-gamma subunit was checked by hemolytic overlay gels and by second dimension SDS-PAGE and immunoblotting. Immunochemical evaluation of C8 alpha-gamma using this system revealed about 5-14% free C8 alpha-gamma in sera with normal C8 and higher levels, from 33-71%, in the C8 beta D heterozygous sera. Functional analysis confirmed the substantial increase of free C8 alpha-gamma in the heterozygous group. We conclude that the C8 in C8 beta D heterozygous sera is characterized by increased amounts of free C8 alpha-gamma due to reduced concentrations of the C8 beta subunit. This finding may help to identify individuals heterozygous for C8 beta deficiency.

Human Pulmonary Surfactant Protein (SP-A), a Protein Structurally Homologous to C1q, Can Enhance FcR- and CR1-mediated Phagocytosis

C1q, a subunit of the first component (C1) of the classical complement pathway, and the pulmonary surfactant protein SP-A are structurally homologous molecules, each having an extended collagen-like domain contiguous with a non-collagenous domain. It is the collagen-like region of C1q that binds to mononuclear phagocytes and mediates the enhancement of phagocytosis of opsonized particles by these cells. Because SP-A enhances the endocytosis of phospholipids by alveolar type II cells and alveolar macrophages, we examined whether these two molecules were functionally interchangeable. The phagocytosis of sheep erythrocytes opsonized with IgG or with IgM and complement was enhanced by the adherence of monocytes or macrophages, respectively, to SP-A. The enhanced response was dependent on the concentration of SP-A used for coating the surfaces, similar to that seen when monocytes were adhered to C1q-coated surfaces. Both the percentage of cells ingesting the opsonized targets and the number of targets ingested per cell increased with increasing concentrations of SP-A. No such enhancement was seen with cells adhered to albumin, iron-saturated transferrin, or uncoated surfaces. However, SP-A did not substitute for C1q in the formation of hemolytically active C1. C1q did not stimulate lipid uptake by alveolar type II cells or alveolar macrophages and had only a slight inhibitory effect on the binding of SP-A to alveolar type II cells. Thus, these results suggested that a function which requires interactions of both the collagenous and the non-collagenous regions (i.e. initiation of the classic complement cascade) could not be mimicked by a protein sharing structural macromolecular similarity but lacking sequence homology in the non-collagen-like region. However, SP-A could substitute for C1q in stimulating a function previously shown to be mediated by the collagen-like domains of the C1q molecule.

Deficiency of the eighth component of complement. Evidence for linkage of C8 alpha-gamma pattern with C8 beta deficiency in sera of twelve patients.

The C8 alpha-gamma subunit of the eighth component of complement was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting in sera from 68 normal individuals, 12 C8 beta-deficient patients (from seven unrelated families), and 10 of the parents of the latter. Three different forms of the C8 alpha-gamma subunit were observed: 34/68 normal individuals were found to have a C8 alpha-gamma triple band (termed C8 alpha-gamma 1, C8 alpha-gamma 2, C8 alpha-gamma 3 variants), 23/68 the C8 alpha-gamma 2 and C8 alpha-gamma 3 variants, and 11/68 the C8 alpha-gamma 1 and C8 alpha-gamma 3 variants. In contrast, all C8 beta-deficient patients had detectable C8 alpha-gamma 2 and C8 alpha-gamma 3 variants but lacked the C8 alpha-gamma 1 variant in addition to the C8 beta subunit. Three out of ten parents of the C8 beta-deficient patients were found to have the C8 alpha-gamma triple band, whereas 7/10, like their children, had the C8 alpha-gamma 2 and C8 alpha-gamma 3 variants only. We conclude that there is a linkage between the C8 alpha-gamma pattern and C8 beta deficiency. These data may support earlier findings that in humans the genes encoding for C8 alpha-gamma and C8 beta are closely linked on chromosome 1.

Endothelial Cells and Bacterial Infection

Endothelial cells are recognized as primary targets of vascular damage in septicemia both as a result of direct action of bacterial toxins or from indirect actions mediated by other cell types and their products (including cytokines, enzymes, and oxygen radicals). We have now demonstrated that endothelial cells phagocytize bacteria 1 Ryan US Phagocytic properties of endothelial cells. in: Ryan US Endothelial cells (vol 3). CRC Press, Boca Raton, Fla1988: 33-49 Google Scholar and are capable of bactericidal activity involving generation of oxygen free radicals. 2 Ryan US Vann JM Endothelial cells: a source and target of oxidant damage. in: Simic MG Taylor KA Ward AF Oxygen radicals in biology and medicine. Plenum Publishing, New York1987: 963-968 Google Scholar We have examined mechanisms involved in recognition of bacterial strains, the mechanisms involved in signal transduction leading to the respiratory burst, and the molecular mechanisms controlling the release of reactive oxygen species. 3 Ryan US, Schultz DR, Goodwin JD, Vann JM, Selvaraj MP, Hart MA. Role of Clq in phagocytosis of S. minnesota by pulmonary endothelial cells, (in press) Google Scholar Endothelial cells distinguish between the mutant (Re), which is taken up in large numbers, and the wild (S) form, which is not phagocytized, of Salmonella minnesota. The Re mutant binds Clq (subunit of the first component of complement), while the S type does not. When bacteria or endothelial cells were incubated with C1q-enriched or C1q-depleted sera, data show that Clq mediates binding to the Re mutant by the globular head region of the molecule and to receptors on the endothelial surface via the collagen-like tail region. Further, the release of superoxide anion in response to phagocytosis appears to involve Clq as a stimulus. 3 Ryan US, Schultz DR, Goodwin JD, Vann JM, Selvaraj MP, Hart MA. Role of Clq in phagocytosis of S. minnesota by pulmonary endothelial cells, (in press) Google Scholar Thus, Clq acts as an opsonin.

Subunit Associations in the Mammalian Pyruvate Dehydrogenase Complex: Structure and Role of Protein X and the Pyruvate Dehydrogenase Component Binding Domain of the Dihydrolipoyl Transacetylase Component

Abstract We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.

Biosynthesis of the 7-mercaptoheptanoic acid subunit of component B [(7-mercaptoheptanoyl)threonine phosphate] of methanogenic bacteria

Deuterium- and {sup 13}C-labeled precursors were used to establish the pathway for the biosynthesis of the 7-mercaptoheptanoic acid moiety of component B in methanogenic bacteria. The extent and position of the label incorporated into 7-mercaptoheptanoic acid were measured from the molecular and fragment ions in the mass spectrum of the methyl ester methylthiol derivative of the 7-mercaptoheptanoic acid. Deuterium from (2,2,2-{sup 2}H{sub 3})acetate was found to be incorporated into four separate positions of 7-mercaptoheptanoic acid. One deuterium was equally distributed between the C-2 and the C-3 of the 7-mercaptoheptanoic acid, and the remaining three were at carbons 4-6. The extent of incorporation of the C-2 and C-3 positions was the same as that observed for the incorporation of (2,2,2-{sup 2}H{sub 3})acetate into the {alpha}-ketoglutarate produced by the cells. (1,2-{sup 13}C{sub 2})Acetate was incorporated into four separate sites of the 7-mercaptoheptanoic acid molecule. On the basis of this and additional information, it is concluded that 7-mercaptoheptanoic acid is biosynthesized from {alpha}-ketosuberate, which arises from {alpha}-ketoglutarate by repeated {alpha}-keto acid chain elongation. The mechanism for the conversion of {alpha}-ketosuberate to a thiol appears to be analogous to that for the conversion of sulfopyruvate to coenzyme M (2-mercaptoethanesulfonic acid).

C1q enhances the phagocytosis of Cryptococcus neoformans blastospores by human monocytes.

We investigated whether C1q, a subunit of the first component of C, could modulate human peripheral blood monocyte-mediated phagocytosis of Cryptococcus neoformans (CN). Adherence of monocytes to C1q-coated surfaces induced a significant enhancement of ingestion of CN blastospores that had been opsonized with specific anticapsular IgG (IgG-CN). Additionally, C1q enhanced the monocyte-mediated phagocytosis of CN opsonized with C (CN-absorbed, nonimmune, normal human serum; C-CN). Ingestion of IgG- and C-CN by control and C1q-stimulated monocytes was maximal by 1 h of incubation. The monocyte-mediated enhancement of phagocytosis caused by C1q was paralleled by a proportionate increase in fungicidal activity, an effect which was maximal by 3 h of incubation. Human serum albumin-adherent, control monocytes exhibited only a low level of killing after 3 h of incubation. C1q enhancement was blocked by preincubation of the surfaces with a goat, polyclonal F(ab')2 anti-C1q. This study describes a new cellular function for the cell surface C1q receptor: the enhancement of phagocytosis of a pathogenic organism by monocytes.

Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase.

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.

Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum

Cells of Methanobacterium thermoautotrophicum were fixed with glutaraldehyde, sectioned and labeled with antibodies against the β subunit of component C (=methyl-CoM reductase) of methyl-CoM reductase system and with colloidal gold-labeled protein A. It was found that the gold particles were located predominantly in the vicinity of the cytoplasmic membrane, when the cells were grown under conditions where methyl-CoM reductase was not overproduced. This finding confirms the recent data obtained with Methanococcus voltae showing via the same immunocytochemical localization technique that in this organism methyl-CoM reductase is membrane associated.

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages.

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.

C1q receptor

Clq is a collagen-like, structurally complex, cationic glycoprotein consisting of 18 similar but distinct polypeptide chains. Each chain consists of an 81-amino-acid-long, collagen-like N-terminal portion and a C-terminal globular region of 136 amino acids. In normal plasma Clq circulates as a subunit of the first component of complement, C1, which is a Ca2+-dependent pentamolecular complex with a structural formula of ClqClr2C1s2. The chapter describes assay methods of Clq. Most of the assays for ClqR used in laboratory are essentially based on a single parameter, i.e., the ability of the receptor to bind to the collagenous region of the Clq molecule to which Clr2Cl s2 complex is also believed to bind. Such a binding invariably inhibits the capacity of Clq to mediate the following functions: rosette formation, association with Clr2Cls2 to form C1 and C1q-dependent cellular cytotoxicity. Another property of the CIqR, which is similar to that of the plasma CIq inhibitor, is its ability to precipitate Clq on solid or liquid medium in a manner comparable to that of antigen-antibody interaction. These properties have been exploited to develop simple assays, some of which are described in the chapter.

Requirement of the nickel tetrapyrrole F430 for in vitro methanogenesis: reconstitution of methylreductase component C from its dissociated subunits.

The subunits and the nickel tetrapyrrole factor F430 of the methylreductase component C from Methanobacterium thermoautotrophicum have been separated from one another and reassociated to form an intact, active enzyme. The individual subunits were extracted from polyacrylamide gels after NaDodSO4/polyacrylamide gel electrophoresis and reassociated with F430 under a H2 atmosphere at 55 degrees C. When these components were reassociated in the presence of the substrate 2-(methylthio)ethanesulfonic acid, 72% of the original activity was recovered. When F430 was omitted, no activity was detected in the reconstituted methylreductase reaction. Individual subunits were inactive, when incubated in the presence of factor F430, 2-(methylthio)ethanesulfonic acid, magnesium acetate, and ATP. Evidence suggests that F430 binds to the large Mr 68,000 subunit of component C.