Search PubMed⌕ Search

Biomedical subjects

S Paul

Publications and source records attributed to S Paul.

At least 181 records · Page 10Linked to original sources
PubMed1994

Molecular cloning of a proteolytic antibody light chain.

The cDNA for an antibody light chain raised by immunization against vasoactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoretic homogeneity. The light chain catalyzed the hydrolysis of VIP efficiently owing to its comparatively high affinity for the substrate. In control experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchange and gel-filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light chain antibody. The amide bond linking methylcoumarinamide (MCA) and arginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (kcat/Km). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively by the alternate substrate, VIP. The Ki and Km values for VIP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling suggested the presence of a serine protease-like site in the light chain. This was supported by inhibition of the hydrolytic activity by serine protease inhibitors, but not by inhibitors of other classes of proteases. These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen recognition function of the light chain combining site.

Amino Acid Sequence↗
PubMed1994

Proteolytic activity of an antibody light chain.

The light chain (L chain) of a mAb raised against unactivated vasoactive intestinal peptide (VIP) hydrolyzed this peptide, whereas the heavy chain (H chain) and an irrelevant L chain were without activity. The reaction kinetics were consistent with efficient substrate recognition by the anti-VIP L chain compared with conventional proteases. The L chain cleaved four peptide bonds clustered between residues 16 and 21 in VIP. Mixtures of the L chain with its H chain partner displayed reduced hydrolytic activity compared with the free L chain, suggesting that the H chain is a modulator of the catalytic activity. These observations suggest: 1) the immune system can generate catalytic sites in the L chain subunit of Abs found in response to polypeptide Ags, and 2) free L chains found in vivo could display an Ag-specific catalytic function.

Amino Acid Sequence↗
PubMed1994

Inhibition of calmodulin-dependent myosin light-chain kinase by growth-hormone-releasing factor and vasoactive intestinal peptide.

In view of the ability of calmodulin to bind vasoactive intestinal peptide (VIP) and growth-hormone-releasing factor (GRF) with high affinity [Stallwood, Brugger, Baggenstoss, Stemmer, Shiraga, Landers and Paul (1992) J. Biol. Chem. 267, 19617-19621], the effects of these neuropeptides on a model calmodulin-dependent enzyme, myosin light-chain kinase (MLCK), were studied. Both peptides were potent inhibitors of MLCK activity. The inhibition of enzyme activity by VIP and GRF was progressively overcome with increasing calmodulin concentrations, with no inhibition observed at a saturating calmodulin concentration. Nanomolar concentrations of MLCK blocked the formation of calmodulin-[125I-Tyr10]VIP complexes. These data provide support for a functional role of VIP and GRF binding by calmodulin.

Amino Acid Sequence↗
PubMed1994

Partitioning of vasoactive intestinal polypeptide into lipid bilayers.

Incubation of radiolabeled vasoactive intestinal polypeptide (VIP) with preformed lipid vesicles composed of phosphatidylcholine, phosphatidylglycerol and cholesterol resulted in reversible and saturable association of the peptide with the lipid bilayer. The pH-optimum for the reaction was in the physiological range. The vesicle-associated peptide displayed enhanced stability to proteolytic digestion, it was efficiently released into the supernatant by detergent-solubilization of the vesicles but remained vesicle-associated during treatment with agents that disrupt ionic interactions. Peptide binding by electrically neutral vesicles was lower than that by negative vesicles. Electron spin resonance studies with 5-doxylstearic acid or 16-doxylstearic acid labeled vesicles suggested that VIP decreased the fluidity close to the surface of the bilayer and increased the fluidity in its hydrophobic core. These observations suggest that VIP can bind and penetrate lipid bilayers.

Amino Acid Sequence↗
PubMed1994

Antigen recognition by an antibody light chain.

A monoclonal antibody to vasoactive intestinal polypeptide (VIP) was reduced and alkylated and its light and heavy chains were purified by denaturing gel filtration. Following renaturation, the light chain displayed sequence-specific binding of VIP. The specific VIP binding activity of several fractions spanning the light chain peak recovered from the gel filtration column was constant, the light chain was electrophoretically homogeneous, the VIP binding activity was precipitated by anti-light chain antibody but not anti-heavy chain antibody and the activity remained associated with a light chain fraction recovered by resolutive chromatography on a hydroxylapatite column. N-terminal amino acid sequencing of the light and heavy chain fractions confirmed the purity of these proteins and suggested that the VL and VH regions belonged to kappa-family II and gamma-family III, respectively. The VIP-binding affinity of the light chain was only 5-fold lower than that of the parent antibody and the light chain did not bind unrelated peptides. These observations suggest that light chains display structural characteristics necessary for high affinity antigen binding.

Amino Acid Sequence↗