| Description | Protease S. aureus, V8 (endoproteinase-Glu-C) specifically cleaves peptide bonds on the COOH-terminal side of either aspartic or glutamic acids (Drapeau et al. 1972). Houmard and Drapeau (1972) report that in the presence of ammonium buffers the enzyme specificity can be limited to glutamoyl bonds. Protease S. aureus, V8 (endoproteinase-Glu-C) specifically cleaves peptide bonds on the COOH-terminal side of either aspartic or glutamic acids (Drapeau et al. 1972). Houmard and Drapeau (1972) report that in the presence of ammonium buffers the enzyme specificity can be limited to glutamoyl bonds. Its rather unique specificity, which can be considered as opposite to that of trypsin, makes it a useful tool for protein chemistry and peptide mapping studies (Cleveland et al. 1977) and (Hall et al. 1978).Characteristics of Protease from S. aureus, V8:Characteristics of Protease, Staph aureus (Endoproteinase Glu-C)Molecular Weight27,000 (Drapeau 1978).Optimal pH4.0 and 7.8 with hemoglobin substrate. (Drapeau et al. 1972).Extinction Coefficientextinction coefficient = 4.26 (Houmard 1976).InhibitorsDiisopropyl fluorophosphate (DFP) and monovalent anions such as F-, Cl-, Br-, CH3COO-, and NO3- (Houmard 1976).MethodEnzyme activity is determined by the casein digestion assay described by Drapeau (1976). Since substrate grade caseins can vary from lot to lot and according to the manner produced, the standardization of casein digestion assays has been difficult to achieve. The use of a reference enzyme preparation is suggested. One unit is that amount of enzyme which releases acid soluble fragments equivalent to 0.001 A280 per minute at 37°C and pH 7.8 under the specified conditions.Reagents1% Casein in 0.05 M Tris⋅PO4 buffer, pH 7.8. (Dissolve 1 gram "Hammersten" casein in 50 ml 0.01 N NaOH with gentle heating and stirring. Add 40 ml reagent grade water and 5.0 ml 1.0 M Tris. Adjust pH to 7.8 with H3PO4 and q.s. to 100 ml.)10% Trichloroacetic acid (TCA)EnzymeDissolve at 1 mg/ml in reagent grade water.ProcedureEquilibrate a series of tubes with 5.0 ml of 1% casein at 37°C for 5 minutes. At zero time add 10 ul or 20 ul of enzyme. Mix. Include a reagent blank. Exactly ten minutes after adding sample, stop reaction by adding 5.0 ml TCA. Mix. Allow tubes to stand ten minutes and then filter. Read A280 of the filtrate.Calculation... Read More | Protein Purity>95% by SDS-PAGEExtinction Coeff.A276 nm = 0.456 at 1.0 mg/mLMolecular Weight8,759 Da (single chain)General DescriptionNatural human C4a is prepared by cleavage of human C4 protein by human C1s. It is produced during activation of both the classical and lectin pathways of complementProtein Purity>95% by SDS-PAGEExtinction Coeff.A276 nm = 0.456 at 1.0 mg/mLMolecular Weight8,759 Da (single chain)General DescriptionNatural human C4a is prepared by cleavage of human C4 protein by human C1s. It is produced during activation of both the classical and lectin pathways of complement. C4a is a member of the anaphylatoxin family of three proteins (C3a, C4a and C5a) produced by the activation of complement (Hugli, T.E. et al. (1981)). It is an unglycosylated polypeptidecontaining 77 amino acids with a molecular mass of 8,759 daltons. Many of the biological functions of C4a are similar to those of C3a, but the specific activities are far below those of C3a. C4a activity is so low, in fact, that it was initially thought to be inactive. These measured activities include inducing muscle contraction in the guinea pig ileum test (spasmogenic activity), desensitization of muscle to C3a stimulation suggesting that the same receptor for both C3a and C4a is involved (tachyphylactic activity) and inducing vascular permeability in human skin (Gorski J.P. et al. (1979)). C4a does not show tachyphylactic activity against C5a or chemotactic activity. Removal of the C-terminal arginine by serum carboxypeptidase N destroys all these activities (Meuller-Ortiz, S.L., et al. (2009)). C4a appears to act through the C3a receptor (C3aR) which is a G-protein coupled receptor found widely distributed on peripheral tissues, lymphoid cells (neutrohphils, monocyes, and eosinophils) and in the central nervous system (astrocytes, neurons and glial cells) (Law, S.K.A. and Reid, K.B.M. (1995)). Physical Characteristics & StructureMolecular weight: 8,759 calculated molecular mass. Observed mass (MALDI-TOF) is 8,762 + 9 mass units. pI = 9.0 to 9.5 (Gorski, J.P. et al. (1981))Amino acid sequence (77 amino acids): NVNFQKAINE KLGQYASPTA KRCCQDGVTR LPMMRSCEQR AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQRC4a is thought to be structurally very similar to C3a and C5a to which it is homologous. Thus its 3D structure is probably similar to the X-ray-derived crystal structureof C3a (Huber, R. et al. (1980)) and the NMR derived structure of C3a: Nettesheim, D.G. et al. (1988); Murray, I. et al. (1999).FunctionSee General Description above. C4a exhibits much weaker biological activities than C3a and C5a. Its activity in inducing erythema and edema in human skin is 25,000-fold weaker than that of C5a and 100-fold weaker than C3a per nanomole. The spasmogenic activity of C4a is 2000-fold weaker than C5a and 100-fold weaker than that of C3a. Due to these differences the role of C4a in these responses in vivo is thought to be negligible.AssaysTwo well established assays for C4a and C3a functional activities include induction of contraction in the guinea pig ileum and the permeation of a dye such as trypan blue from the vasculature into skin. The anaphylatoxins also induce mast cell degranulation, (measured as histamine release), platelet aggregation, IL-1 release from monocytes and the release of prostaglandins and leukotrienes from many cells and tissues. The other assays used for C3a (Dodds, A.W. and Sim, R.B. (1997)) should also respond to C4a, but few reports have described utilizing these assays with C4a. ELISA kits for the assay of C4a levels (or more correctly C4a desArg levels) in blood and other fluids are sold by several companies. These measurements are useful for detecting complement activation in vivo, but the interpretation of their meaning is complicated by the fact that clearance of the anaphylatoxins is rapid. In vivoFreshly drawn normal human serum contains significant levels of all three anaphylatoxins. Although these may represent the resting concentration in vivo it is difficult to draw or store blood without some complement activation so a true in vivo concentration is difficult to determine. The presence of EDTA and Futhan in the collection tubes can minimize this background (Pfeifer, P.H. et al. (1999)). Full activation of all C4 in blood (600µg/mL) would result in ~3,400 nM C4a (~30 µg/mL). Due to the low biological activity of C4a it could require activation of most of the C4 in a small region to achieve the micromolar C4a concentrations necessary to elicit a response.RegulationC4a levels are regulated by three processes: formation, inactivation and clearance. There are two enzymes that cleave C4 and release C4a: C1s and MASP-2. C4a is “inactivated” by removal of its C-terminal arginine amino acid. The product C4a desArg (or C4a without the C-terminal arginine) is produced by the action of the plasma enzyme carboxypeptidase N (Mueller-Ortiz S.L. et al. (2009)). The inactivation is rapid and most C4a is converted to C4a desArg within minutes of its formation. Inactivated C4a lack measurable biological activity. Because of the large number of cells bearing C3a/C4areceptors (endothelial, immune, smooth muscle, neuronal, etc.) the capture, internalization and digestion of C4a and C4a desArg probably results in its removal from circulation.DeficienciesA deficiency of C4 or a deficiency of all of the enzymes that cleave C4 to generate C4a could result in the absence of C4a. There are no known complete deficiencies of all ofthe C4 cleaving enzymes. Examples of C4 deficient humans and mice exist (Wessels, M.R. et al. (1995)), but the degree to which pathologies associated with C4 deficiency are due to the lack of C4 or the absence of C4a is unclear. DiseasesThere are no known diseases connected to C4a or C4a desArg. Precautions/Toxicity/HazardsThe source of C4a is human serum, therefore appropriate precautions must be observed even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.Injection can cause anaphylatic shock which is a generalized circulatory collapse similar to that caused by an allergic reaction.Hazard Code: B WGK Germany 3... Read More | Proteinase K is a stable and highly reactive serine protease. Evidence from crystal and molecular structure studies indicates the enzyme belongs to the subtilisin family with an active-site catalytic triad (Asp39-His69-Ser224). It is stable in a broad range of environments: pH, buffer salts, Proteinase K is a stable and highly reactive serine protease. Evidence from crystal and molecular structure studies indicates the enzyme belongs to the subtilisin family with an active-site catalytic triad (Asp39-His69-Ser224). It is stable in a broad range of environments: pH, buffer salts, detergents (SDS), and temperature. In the presence of 0.1-0.5% SDS, proteinase K retains activity and will digest a variety of proteins and nucleases in DNA preparations without compromising the integrity of the isolated DNA.ApplicationUseful for the proteolytic inactivation of nucleases during the isolation of DNA and RNA.Removes endotoxins that bind to cationic proteins such as lysozyme and ribonuclease A.Reported useful for the isolation of hepatic, yeast, and mung bean mitochondriaDetermination of enzyme localization on membranesTreatment of paraffin embedded tissue sections to expose antigen binding sites for antibody labeling.Digestion of proteins from brain tissue samples for prions in Transmissible Spongiform Encephalopathies (TSE) research... Read More | Inquire | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：p53 is well known for its key role as a tumor suppressor protein. It is 393 amino acids (aa) in length with a predicted molecular weight of 44 kDa. It belongs to the p53 family that also includes p63 and p73Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：p53 is well known for its key role as a tumor suppressor protein. It is 393 amino acids (aa) in length with a predicted molecular weight of 44 kDa. It belongs to the p53 family that also includes p63 and p73. Structurally, p53 is characterized by an N-terminal transactivation domain, central DNA-binding and oligomerization domains, and a C-terminal regulatory domain. It is thought to exist as a homotetramer, and it exhibits approximately 72% and 76% aa identity with its mouse and rat orthologs, respectively. Mutations in the p53 gene are one of the most frequent genomic events accompanying oncogenic transformation. p53 responds to signals such as DNA damage or cell stress primarily through its actions as a transcription factor. Among its gene targets are a range factors that promote DNA repair mechanisms or apoptosis, including cell cycle regulatory proteins and members the Bcl-2 family. Because of its critical role in genomic homeostasis, p53 activities are tightly regulated by a network of protein-protein interactions, microRNAs, and a range of post-translational modifications, including phosphorylation, acetylation, methylation, and ubiquitination. A widely studied regulator is Murine Double Minute 2 (MDM2). MDM2 is known to suppress p53 activity through direct binding or through its actions as a Ubiquitin ligase (E3) that catalyzes p53 ubiquitination and proteasome-mediated degradation... Read More |