Journal of Protein Chemistry

The inactivation and conformational changes of the bacterial chaperonin GroEL have been studied in SDS solutions with different concentrations. The results show that increasing the SDS concentration caused the intrinsic fluorescence emission intensity to increase and the emission peak to slightly blue-shift, indicating that increasing the SDS concentration can cause the hydrophobic surface to be slightly buried. The changes in the ANS-binding fluorescence with increasing SDS concentration also showed that the GroEL hydrophobic surface decreased. At low SDS concentrations, less than 0.3 mM, the GroEL ATPase activity increased with increasing SDS concentration. Increasing the SDS concentration beyond 0.3 mM caused the GroEL ATPase activity to quickly decrease. At high SDS concentrations, above 0.8 mM, the residual GroEL ATPase activity was less than 10% of the original activity, but the GroEL molecule maintained its native conformation (as indicated by the exposure of buried thiol groups, electrophoresis, and changes of CD spectra). The above results suggest that the conformational changes of the active site result in the inactivation of the ATPase even though the GroEL molecule does not markedly unfold at low SDS concentrations.

A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 → 3)-β-d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.

Purified human glucocerebrosidase isolated from placenta was modified with [14C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed byα-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [14C]-carboxymethylated-cysteine identified as CM-Cys18. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys18. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 andα-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced withβ-mercaptoethanol followed by S-carboxymethylation with [14C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys4-Cys16, Cys23-Cys342, and Cys126-Cys248. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of VL and VH regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [14C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS–PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.

β-Bungarotoxin (β-Bgt), the main presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and B chain, cross-linked by an interchain disulfide bond. The A and B chain cDNAs were subcloned into expression vectors pT7-7 and pET20b(+), respectively, and transformed into Escherichia coli strain BL21(DE3). The expressed protein was isolated from the inclusion bodies of E. coli and subjected to refolding into its folded structure. The yields of the refolded A and B chains increased markedly by at least 100-fold after substituting Ser for Cys15 of A chain and Cys55 of B chain, which formed an interchain disulfide bond. Either the A(C15S) chain or B(C55S) chain alone or in combination cannot exhibit the phospholipase A2 activity or synaptosome binding activity of β-Bgt. Nevertheless, the results of competitive enzyme-linked immunoassay, CD spectra, and fluorescence measurement revealed that the A(C15S) chain and B(C55S) chain possessed a native-like structure like the subunits of native β-Bgt. Moreover, the interfacial interaction between the A and B chains explored by glutaraldehyde cross-linking revealed the essential aspects of the intact interchain disulfide bond in this interaction. This suggests that the formation of the interchain disulfide bond should not be a crucial step for the formation of folded A and B chains in the venom glands, and that the integrity of the interchain disulfide linkage favors the subunit interaction that consequently fulfills the functional mechanism of β-Bgt.

The quaternary structure of ATP-dependent phosphoenolpyruvate (PEP) carboxykinases is variable. Thus, the carboxykinases from Escherichia coli, Trypanosoma brucei, and Saccharomyces cerevisiae are monomer, homodimer, and homotetramer, respectively. In this work, we studied the effect of temperature on the stability of the enzyme activity of these three carboxykinases, and have found that it follows the order monomer > dimer > tetramer. The inactivation processes are first order with respect to active enzyme. The presence of substrates leads to an increase in the thermal stability of all three PEP carboxykinases. The protection effect of the substrates on the thermal inactivation of these enzymes suggests similarities in the substrate-bound form of these proteins. We propose that the higher structural complexity of some PEP carboxykinases could be related to the acquisition of properties of relevance in vivo.

Bovine antithrombin (ATIII) is a glycoprotein of Mr 56,600. Its primary structure was established using peptide sequences from five different digests. Bovine ATIII exhibits four glycosylation sites as well as human ATIII. The primary structures of bovine and human ATIII were compared: all the residues required for the integrity of the heparin-binding domain are strictly conserved. However, there are differences in the secondary structures of both proteins, bovine and human ATIII.

The aluminum and yeast hexokinase interaction was studied. Structural changes were correlated with variations in protein functionality. Results show two different behaviors: At low metal concentrations preferential adsorption of metal (and water exclusion) induces aggregate formation. No significant changes in the protein structure occur, but there is a continuous loss of activity (from the first concentration). At large salt concentrations a monomerization process and a conformational change in the secondary structure as well as in the three-dimensional structure take place. This change reduces the percentage of α-helix conformation, gives thermal stability to the protein, and allows the exposure of some tryptophan residue and hydrophobic regions. The protein inhibition increases. Conformational change and monomerization may allow access of the metal to the substrate site, mainly the ATP site. The inhibition in any case is of mixed type with a competitive component.