4-thiopyridine and 4-4--dipyridyl-disulfide

Recombinant human gastric lipase (rHGL) and three of its cysteine mutants (cysteine 227, 236, and 244 substitued for threonine or serine) were expressed in the baculovirus/insect cell system and purified to homogeneity by performing a two-step procedure. Substituting Ser for Cys 227 and Cys 236 resulted in mutant lipases with a significantly lower level of activity (30% and 22%, respectively) on a short chain triglyceride (tribuyrin) substrate, while the mutation at position 244 only slightly reduced the activity. Using 4, 4'-dithiopyridine (4-PDS) as a sulfhydryl reagent on the above mutants, it was possible to clearly identify the single sulfhydryl residue at position 244 and consequently, the disulfide bridge at position 227-236. No potential disulfide bridges were formed during the protein folding between cysteines 227-244 or between cysteines 236-244, as thought to occur in the case of rabbit gastric lipase (RGL). The present results are consistent with the recently determined 3D-structure of rHGL.

Topics: Amino Acid Substitution; Animals; Baculoviridae; Catalysis; Cell Line; Cysteine; Disulfides; Gastric Mucosa; Humans; Insecta; Kinetics; Lipase; Protein Folding; Pyridines; Recombinant Proteins; Sodium Dodecyl Sulfate; Titrimetry; Triglycerides

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cattle; Cysteine; Cytoplasm; Darkness; Disulfides; Light; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidation-Reduction; Peptide Fragments; Protein Structure, Secondary; Protein Structure, Tertiary; Pyridines; Rhodopsin; Sulfhydryl Reagents; Transducin

2,2'-Dithiodipyridine reacts rapidly with sheep liver cytoplasmic aldehyde dehydrogenase in the presence of NAD +, resulting in activation of the enzyme by 2 to 2.5-fold (when assayed in the usual way). This is followed by the slow loss of most of the enzyme activity during the next few hours at 25 degrees C. 2-Thiopyridone is displaced from the labeled enzyme at approximately the same rate as activity is lost. This is explained in terms of the initial modification of an enzymatic thiol group (giving activation) followed by the reaction of the labeled group with a second enzymatic thiol group, resulting in the formation of a disulfide bond and the inactivation of the enzyme. 4,4'-Dithiodipyridine reacts in a broadly similar way, although both the loss of label and loss of activity are faster and do not correlate with each other as well as for the 2,2' isomer. The results suggest that the dithiodipyridines act to produce the same enzymatic disulfide bond as has been shown to arise from the reaction of the enzyme with disulfiram (a drug used in alcoholism treatment). The implications of the results are discussed with reference to the proposed mechanism of action of aldehyde dehydrogenase. It is concluded that the thiol group initially modified by disulfiram is unlikely to be catalytically essential to the dehydrogenase action of the enzyme.

Topics: 2,2'-Dipyridyl; Aldehyde Dehydrogenase; Animals; Cytoplasm; Disulfides; Disulfiram; Enzyme Activation; Kinetics; Liver; NAD; Pyridines; Sheep