Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Complement C1r”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources
PubMed1980

Analysis of receptor-mediated C1q binding to human peripheral blood mononuclear cells.

The binding of C1q to human peripheral blood leukocytes has been investigated. Studies with fluorescein conjugated F(ab')2 anti-C1q show that few (0 to 4%) normal leukocytes isolated in the presence of EDTA have C1q on their surface. However, approximately 26% of the mononuclear cell population is able to bind added C1q. Quantitative binding studies using 125I-C1q show that the binding to mononuclear cells is specific, saturable, and reversible. Scatchard plot analyses indicate an approximate equilibrium constant of 1.2 times 10(7) M-1. C1q binding appears to be mediated via the collagenous portion of the molecule in that 1) type I collagen inhibits this binding; 2) C1 reconstituted from purified C1q, C1r, and C1s does not bind to mononuclear cells, whereas the same amount of free C1q binds avidly; and 3) C1q enhances the binding of aggregated IgG to mononuclear cells.

Binding Sites↗
PubMedDate not supplied

[Role of C1 subcomponents in platelet aggregation induced by aggregated IgG].

Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA-treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1s or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG. The possible association between the different C1 subcomponents and human platelets is discussed.

Complement C1↗
PubMed1976

C1 and human platelets. III. Role of C1 subcomponents in platelet aggregation induced by aggregated IgG.

Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1r or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG.The possible association between the different C1 subcomponents and human platelets is discussed.

Complement C1↗
PubMed2001

Beta-amyloid fibrils activate the C1 complex of complement under physiological conditions: evidence for a binding site for A beta on the C1q globular regions.

Previous studies based on the use of serum as a source of C have shown that fibrils of beta-amyloid peptides that accumulate in the brain of patients with Alzheimer's disease have the ability to bind C1q and activate the classical C pathway. The objective of the present work was to test the ability of fibrils of peptide Abeta1-42 to trigger direct activation of the C1 complex and to carry out further investigations on the site(s) of C1q involved in the interaction with Abeta1-42. Using C1 reconstituted from purified C1q, C1r, and C1s, it was shown that Abeta1-42 fibrils trigger direct C1 activation both in the absence of C1 inhibitor and at C1 inhibitor:C1 ratios up to 8:0, i.e., under conditions consistent with the physiological context in serum. The truncated peptide Abeta12-42 and the double mutant (D7N, E11Q) of Abeta1-42 did not yield C1 activation, providing further evidence that the C1 binding site of beta-amyloid fibrils is located in the acidic N-terminal 1-11 region of the Abeta1-42 peptide. Binding studies performed using a solid phase assay provided strong evidence that C1q interacts with Abeta1-42 fibrils through its C-terminal globular regions. In contrast to previous studies based on a different experimental design, no significant involvement of the C1q collagen-like domain was detected. These findings were confirmed by additional experiments based on C1 activation and C4 consumption assays. These observations provide direct evidence of the ability of beta-amyloid fibrils to trigger activation of the classical C pathway and further support the hypothesis that C activation may be a component of the pathogenesis of Alzheimer's disease.

Amino Acid Sequence↗
PubMed1987

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.

Antigen-Antibody Complex↗
PubMed2005

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules.

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

Amino Acid Sequence↗
PubMed1986

Activation of the first component of human complement, C1, by monoclonal antibodies directed against different domains of subcomponent C1q.

Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.

Animals↗
PubMed2003

Interaction of the C1 complex of complement with sulfated polysaccharide and DNA probed by single molecule fluorescence microscopy.

The complex C1 triggers the activation of the Complement classical pathway through the recognition and binding of antigen-antibody complex by its subunit C1q. The globular region of C1q is responsible for C1 binding to the immune complex. C1q can also bind nonimmune molecules such as DNA and sulfated polysaccharides, leading either to the activation or inhibition of Complement. The binding site of these nonimmune ligands is debated in the literature, and it has been proposed to be located either in the globular region or in the collagen-like region of C1q, or in both. Using single molecule fluorescence microscopy and DNA molecular combing as reporters of interactions, we have probed the C1q binding properties of T4 DNA and of fucoidan, an algal sulfated fucose-based polysaccharide endowed with potent anticomplementary activity. We have been able to visualize the binding of C1q as well as of C1 and of the isolated collagen-like region to individual DNA strands, indicating that the collagen-like region is the main binding site of DNA. From binding assays with C1r, one of the protease components of C1, we concluded that the DNA binding site on the collagen-like region is located within the stalk part. Competition experiments between fucoidan and DNA for the binding of C1q showed that fucoidan binds also to the collagen-like region part of C1q. Unlike DNA, the binding of fucoidan to collagen-like region involves interactions with the hinge region that accommodate the catalytic tetramer C1r2-C1s2 of C1. This binding property of fucoidan to C1q provides a mechanistic basis for the anticomplementary activity of the sulfated polysaccharide.

Buffers↗
PubMed1980

The first component of complement. I. Purification and properties of native C1.

The first component of complement has been purified by using affinity chromatography on Sepharose-bound IgG. Unlike earlier procedures that yield the activated form of C1, in this method C1 is maintained in the native form by the protease inhibitor p-nitrophenyl, p'-guanidinobenzoate (NPGB). The procedure requires only two steps and yields pure C1 as judged both by SDS-PAGE analysis and by effective molecule calculations. The yields have varied from 30 to 50% in over 50 preparations. The functional properties of the purified native C1 correspond to those of C1 in serum. The dose-response activity profile is nonlinear, but becomes linear when C1 IS ALLOWED TO SELF-ACTIVATE. From SDS-PAGE analysis of the self-activated C1, all the C1r and C1s subcomponents are converted to the activated split products, indicating that all C1 molecules are biologically active. The recovery of C1 activity is dependent on the use of a heterologous source for the IgG on the affinity absorbant. The conditions of binding and elution from the Sepharose-IgG column are critical, indicating that immunoglobulin-bound C1 is rapidly inactivated under physiologic conditions by serum inactivators. The activation of the purified C1 in fluid phase has been explored both in the presence and absence of C1-inhibitor.

Benzoates↗
PubMed1969

Fluid phase destruction of C2hu by C1hu. II. Unmasking by C4ihu of C1hu specificity for C2hu.

It has been demonstrated that C1 isolated in the unactivated form fails to inactivate C4 or C2 in the fluid phase, while the activated molecule, C1 rapidly converts C4 to hemolytically inactive C4i, but does not efficiently inactivate C2. The production and presence of C4i now confers on C1 the ability to rapidly inactivate C2. After heating at 56 degrees C, so as to destroy the hemolytic activity, heat inactivated C1 is still capable of inactivating C4 but the presence of C4i no longer confers an ability to inactivate C2. Studies with the subunits of C1-C1q, C1r, C1s, indicate that the action of C1s on C2 can be inhibited by C1r and that this inhibition is reversed by the presence of homologous C4. These studies indicate that the interaction of C4i with a heat labile receptor conformation in C1 uncovers a masked specificity for C2.

Animals↗
PubMed1991

Synthesis of C1 inhibitor in fibroblasts from patients with type I and type II hereditary angioneurotic edema.

Patients with hereditary angioneurotic edema (HANE) have serum levels of functionally active inhibitor of the first component of complement (C1 INH) between 5 and 30% of normal, instead of the 50% expected from the single normal allele. Increases in rates of catabolism have been documented in patients with HANE and certainly account for some of decrease in C1 INH level. A possible role for a decrease in synthesis of C1 INH in producing serum levels of C1 INH below the expected 50% of normal has not been well studied. We studied the synthesis of C1 INH in skin fibroblast lines, which produce easily detectable amounts of C1 INH. In type I HANE cells, C1 INH synthesis was 19.6 +/- 4.0% (mean +/- SD) of normal, much less than the 50% predicted. In type II HANE cells, the total amount of C1 INH synthesis (functional and dysfunctional) was 98.9 +/- 17% of normal; the functional protein comprised 43% of the total. Thus, type II HANE cells synthesized functional C1 INH at a much greater rate than for the type I cells. In both type I and II HANE cells, amounts of steady-state C1 INH mRNA levels paralleled rates of C1 INH synthesis, indicating that control of C1 INH synthesis occurred at pretranslational levels. Both type I and type II fibroblasts synthesized normal amounts of C1r and C1s. These data suggest that the lower than expected amounts of functionally active C1 INH in type I HANE may be due, in part, to a decrease in rate of synthesis of the protein, and that the expressions of the normal C1 INH allele in HANE is influenced by the type of abnormal allele present.

Adult↗
PubMed1977

Biosynthesis of the first component of complement by human fibroblasts.

1. Haemolytic activity corresponding to that of the first component of complement (C1) was synthesized and secreted by all nine human fibroblast cell lines examined. No activity was found in the culture media of a variety of other human cell lines. 2. The component-C1 haemolytic activity secreted by the fibroblast lines behaved in an identical manner, in most respects, with that of the component-C1 haemolytic activity of human serum. The component-C1 haemolytic activity secreted by fibroblasts, however, was less susceptible to inhibition by rabbit fragment F(ab')(2) anti-(human subcomponent C1q) than was the component-C1 haemolytic activity of human serum. 3. Biosynthesis of fibroblast component-C1 haemolytic activity was inhibited by the presence of cycloheximide and regained on its removal. 4. Incorporation of radioactivity into proteins secreted by the fibroblasts and release of component-C1 haemolytic activity by the fibroblasts both increased in a linear manner until several days after the cultures had reached a state of confluent growth. 5. Radioactivity was incorporated into subcomponents C1q, C1r and C1s, as judged by the formation of specific immunoprecipitates and by absorption with immune aggregates. 6. The immunoprecipitates formed by using antisera against subcomponents C1r and C1s were run on polyacrylamide gels in sodium dodecyl sulphate, and this provided convincing physiochemical evidence for the biosynthesis of these subcomponents de novo. 7. The results obtained with immunoprecipitates formed by using anti-(subcomponent C1q) suggest that subcomponent C1q may be synthesized and secreted by fibroblast cell lines in vitro, in a form with a higher molecular weight than that of subcomponent C1q which is isolated by conventional techniques of protein fractionation from fresh serum.

Cell Line↗
PubMed1994

Inherited complement C3 deficiency: a defect in C3 secretion.

The molecular basis of inherited complement C3 deficiency in a 20-year-old newly diagnosed male patient was studied. Using an enzyme-linked immunosorbent assay, the patient's C3 serum level was found to be approximately 7 micrograms/ml, which is less than 1% of normal. In contrast, Northern analysis indicated that the patient's C3 mRNA was of normal size and quantity. Peripheral blood monocytes (PBM) and skin fibroblast cultures (F) from the patient and from healthy donors were labeled for 2 h with [35S] methionine. Analysis of cell lysates and supernatants by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated normal levels of C3 in lysates of patient's PBM and F. However, C3 secretion in the patient's cells was extremely reduced, with pulse-chase experiments demonstrating a long delay in the disappearance of intracellular C3. Secretion of C1r and factor B by the patient's cells was normal. Lipopolysaccharide and interleukin-1 increased C3 synthesis in the patient's PBM and F, but had no effect on the secretion. SDS-PAGE analysis of trypsin-cleaved intracellular C3 revealed an aberrant cleavage profile for the patient's C3. Collectively, these data indicate that C3 deficiency in this patient is due to a defect in the C3 secretion, probably as the result of abnormality in the proC3 structure.

Adolescent↗
PubMed1998

Baculovirus-mediated expression of truncated modular fragments from the catalytic region of human complement serine protease C1s. Evidence for the involvement of both complement control protein modules in the recognition of the C4 protein substrate.

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (gamma-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP2-ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP2-ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6-3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic gamma-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1s. Likewise, the activated fragments gamma-B, CCP2-ap-SP, and ap-SP retained C1s ability to cleave C2 in the fluid phase. In contrast, whereas fragment gamma-B cleaved C4 as efficiently as C1s, the C4-cleaving activity of CCP2-ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1s has no significant effect on catalytic activity.

Animals↗
PubMed1977

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.

Antibodies, Viral↗
PubMed1987

Cloning and characterization of cDNAs encoding the complete sequence of decay-accelerating factor of human complement.

cDNAs encoding the complement decay-accelerating factor (DAF) were isolated from HeLa and differentiated HL-60 lambda gt cDNA libraries by screening with a codon preference oligonucleotide corresponding to DAF NH2-terminal amino acids 3-14. The composite cDNA sequence showed a 347-amino acid protein preceded by an NH2-terminal leader peptide sequence. The translated sequence beginning at the DAF NH2 terminus encodes four contiguous approximately equal to 61-amino acid long repetitive units of internal homology. The repetitive regions contain four conserved cysteines, one proline, one glycine, one glycine/alanine, four leucines/isoleucines/valines, one serine, three tyrosines/phenylalanines, and one tryptophan and show striking homology to similar regions previously identified in factor B, C2, C4 binding protein, factor H, C1r, factor XIII, interleukin 2 receptor, and serum beta 2-glycoprotein I. The consensus repeats are attached to a 70-amino acid long segment rich in serine and threonine (potential O-glycosylation sites), which is in turn followed by a stretch of hydrophobic amino acids. RNA blot analysis of HeLa and HL-60 RNA revealed three DAF mRNA species of 3.1, 2.7, and 2.0 kilobases. The results indicate that portions of the DAF gene may have evolved from a DNA element common to the above proteins, that DAF cDNA predicts a COOH-terminal anchoring polypeptide, and that distinct species of DAF message are elaborated in cells.

Amino Acid Sequence↗
PubMed1979

Interaction of C1-inhibitor with the C1r and C1s subcomponents in human C1.

1. Insoluble IgG-ovalbumin aggregates were used to bind and activate C1 from human serum. The bound C1 provided a useful reagent for studying the interaction of C1 subcomponents with C1-inhibitor. 2. C1-inhibitor bound to both subcomponents (C1r and C1s in C1 and formed stable complexes of respective apparent molecular weights 197,000 and 185,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The binding reaction proceeded more readily with C1s than with C1r and was correlated with the inhibition of C1s esterase activity. 3. At physiological ionic strength, binding of C1-inhibitor to subcomponents C1r and C1s caused release of these subcomponents from the C1-immune aggregates complex, indicating that C1-inhibitor binding decreased the inter-subcomponent binding forces in C1. At low ionic strength, however, this release did not occur.

Complement C1↗
PubMed1985

C1 dissociation in serum: estimation of free C1q by electroimmunoassay.

A two-stage electroimmunoassay was developed for measuring macromolecular C1 (C1qrs) and free C1q. The method was based on Ca2+ dependent fixation of C1qrs to agarose, followed by immune precipitation of dissociated C1s in the presence of EDTA. Free C1q was estimated from the increase in C1qrs resulting from saturation of C1q in the samples with purified C1r-C1s. The assay system was studied under various experimental conditions. Combined analysis by electroimmunoassay and crossed immunoelectrophoresis indicated that part of the free C1q in undiluted normal serum could be attributed to physiological C1 activation. Owing to concentration dependent C1qrs dissociation the proportion of free C1q increased with the dilution of serum. Results obtained with serum and with purified C1qrs were consistent with the formation of an equimolar C1q:C1r-C1s complex. However, the capacity for C1r-C1s binding appeared to be higher in the purified system than in serum. Serum concentrations of free C1q were high in some of the patients with disease conditions characterized by increased C1 activation, such as systemic lupus erythematosus or primary biliary cirrhosis.

Complement Activating Enzymes↗