Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.
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PMID:A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits. 1 53

Antisera reactive with the subunits of transcarboxylase were produced by injecting rabbits with the 12 SH and 5 SE subunits prepared from trypsin-treated enzyme and with the 12 SH subunit prepared from native transcarboxylase by avidin-Sepharose chromatography. Biotinyl peptides (46 and 65 amino acid residues), released from the enzyme by brief trypsin treatment, formed complexes with avidin and this noncovalent conjugate was used as an antigen to prepare antibodies reactive with the 1.3 SE biotin carboxyl carrier protein. All of these antisera were capable of inhibiting and precipitating intact transcarboxylase. In competitive binding experiments utilizing enzyme inhibition to detect antibody binding, it was found that only the 5 SE subunit was capable of preventing the anti-5 SE sera from inhibiting the enzyme. This technique is useful as a rapid, specific assay for the 5 SE subunit. Both the 5 SE and 12 SH subunits were capable of preventing the anti-12 SH sera from inhibiting the enzyme. To further study this apparent cross-reactivity, the antisera were tested for their capacity to inhibit the partial reactions of transcarboxylase as catalyzed by the isolated subunits. These studies revealed that the anit-12 SH serum inhibits the partial reaction catalyzed by the 12 SH subunit, but preincubation with the 5 SE subunit does not relieve this inhibition, confirming that specific antibody against the 12 SH subunit was formed. These studies also showed that the anti-12 SH serum could inhibit the 5 SE partial reaction, apparently demonstrating cross-reactivity with the 5 SE subunits of the intact enzyme. A panel of immunologic methods was used to characterize the cross-reactivity and quantitate any cross-contamination of the subunit preparations. Passive hemagglutination-hemagglutination inhibition, double immunodiffusion in gels, and competitive precipitation of radioactive antigen all demonstrated cross-reactivity between the isolated subunits and the antisera. Although the subunit preparations apparently are cross-contaminated to a minor extent, this seems insufficient to account for the quantitative aspects of the immunologic cross-reactivity of the subunits with the antisera. Structural homology between the subunits of transcarboxylase is considered to be responsible for the observed cross-reactivity.
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PMID:Immunochemistry of the subunits of transcarboxylase. 82 13

In this paper we show that the native form of transcarboxylase may be a species which has six rather than three subunits attached to the central subunit. We have designated this form as the 26 S enzyme. Electron micrographs support the view that the six subunits are attached in sets of three at the opposite faces of the central subunit, in contrast to the 18 S form in which all three subunits appear to be attached only at one face. In addition, evidence is presented that the dissociation of the 26 S to the 18 S form of transcarboxylase occurs with the loss of three subunits exclusively from one face of the central subunit. This result may indicate that the two faces of the central subunit differ structurally or there is negative cooperativity in the dissociation of subunits. The 26 S transcarboxylase, which was made by attachment of subunits to the 18 S enzyme or trypsin-treated 18 S enzyme was shown to have subunits on both faces of the central subunit. Treatment of the 26 S enzyme with carbodimide to cross-link the subunits to the central subunit and thus stabilize the structure resulted in improved electron micrographs. A model of the 26 S form of the enzyme is presented.
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PMID:Electron microscopy of the large form of transcarboxylase with six attached subunits. 83 26

Transcarboxylase is made up of a central hexameric subunit (S20,W similar 12 S), three peripheral dimeric metallo subunits (S20,W similar to 5 S), and six biotinyl carboxyl carrier subunits (S20,W similar to 1.3 S). The results presented here show that the carboxyl carrier subunit is required for assembly of the 12S and 5S subunits into the oligomer. However, only a portion of the subunit is required for assembly. On treatment of transcarboxylase briefly with trypsin at pH 6.3 extremely susceptible peptide bonds of the carboxyl carrier protein are cleaved releasing biotinyl peptides of about similar to 66 and similar to 40 residues. The resulting trypsinized transcarboxylase, though enzymatically inactive, remains essentially intact as judged by its hydrodynamic and molecular sieving properties. The modified enzyme can be dissociated at pH 8 to the central 12S subunit and peripheral 5S subunit to which the residual portion(s) of the cleaved carboxyl carrier protein is still attached. These components can then be separated by molecular sieving. The residual portion of the carboxyl carrier protein (non-biotinyl peptide) can then be isolated by dissociation of the 5S subunit complex at pH 9 and by chromatography over Bio-Gel A-1.5m. The isolated non-biotinyl peptide has been shown to contain the combining domain of the 1.3SE carboxyl carrier protein since it causes combination of the 12S and 5S subunits. Active enzyme is formed by combination of the intact carboxyl carrier protein and the 12S and 5S subunits and an inactive oligomer of similar size is formed if the non-biotinyl peptide is used in place of the carboxyl carrier protein. The similar to 66- and similar to 40-residue biotinyl peptides, which are released by the trypsin treatment, apparently occur on an exposed portion of the enzyme. This portion of the carboxyl carrier protein apparently serves to place the biotinyl group adjacent to the two substrate sites of the enzyme, one of which is on the peripheral subunit and the other on the central subunit. Thus the carboxyl carrier protein has two functions: one portion holds the 12S and 5S subunits in juxtaposition and the other portion orients the biotinyl group adjacent to the substrate sites so that it may function as a carboxyl carrier between the sites.
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PMID:Isolation of peptides from the carboxyl carrier subunit of transcarboxylase. Role of the non-biotinyl peptide in assembly. 112 90

The results presented here show that isolated subunits of transcarboxylase specifically catalyze the two partial reactions of transcarboxylation as shown in eq 1-3. The 12S central subunit is active in the transcarboxylation with methylmalonyl-CoA but inactive with oxalacetate and the peripheral metallo 5S subunit is active in the transcarboxylation with oxalacetate but inactive with methylmalonyl-CoA. These subunits, likewise, are specific for the reverse partial reactions; the central subunit catalyzing transfer from the carboxylated biotinyl group to propionyl-CoA to yield methylmalonyl-CoA and the peripheral subunit to pyruvate to yield oxalacetate. Thus, the central subunit contains the sites for the CoA esters (methylmalonyl-CoA and propionyl-CoA) and the peripheral metallo subunits for the keto acids (oxalacetate and pyruvate). In the overall reaction the biotinyl carboxyl carrier protein acts as a shuttle to carry the carboxyl groups between the two subunits. Biotin and certain biotin analogs are inactive in these partial reactions but the similar to 40- or similar to 66-residue biotinyl peptides, which are derived from the carboxyl carrier protein, are active. Transcarboxylase can be reconstituted from its isolated subunits and a comparison was made of the rate of the overall reaction when the subunits were assembled, as in the intact enzyme, with that obtained when the reaction was catalyzed by the nonassembled subunits. In the latter case, since the biotinyl carboxyl carrier subunit must diffuse from one subunit to the other, the overall reaction is much slower than with the assembled subunits. The reaction with trypsinized transcarboxylase from which the similar to 66-residue and similar to 40-residue biotinyl peptides have been stripped, likewise, was slow even though the biotinyl peptides were added to the reconstitution mixture. The 12SH and 5SE subunits remain assembled after trypsin treatment but the biotinyl peptides apparently do not combine firmly or properly with the trypsinized enzyme and the biotinyl group apparently must oscillate as a carboxyl carrier between the two sites on the subunits by diffusion.
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PMID:Evidence that the two partial reactions of transcarboxylation are catalyzed by two dissimilar subunits of transcarboxylase. 112 91