Photoaffinity labeling of a peptide substrate to myosin light chain kinase.

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.

Solid-phase synthesis of biologically active lipopeptides as analogs for spirochetal lipoproteins.

Bacterial lipoproteins, which are of particular interest because of their immunomodulatory activities, share a common N-terminal structural motif that consists of an N-acyl-S-diacylglyceryl cysteine residue. Synthetic tripalmitoylated analogs of the N-terminal sequences of several bacterial lipopetides have been found to reproduce the immunological activities of the corresponding intact lipoproteins. Methods for the synthesis of lipopeptide analogs of bacterial lipoproteins have hitherto relied upon the coupling of peptide moieties, lacking the N-terminal cystienyl residue, with a tripalmitoylglyceryl cysteine moiety synthesized separately in solution. A method is described here by which rapid and convenient synthesis of the entire lipopeptide is accomplished by solid-phase methods in which the N-terminal cysteinyl derivative is assembled stepwise while attached to the completed peptide moiety prior to cleavage from the resin. The method has been used to synthesize two lipohexapeptides representing the N-terminal sequences of the 47-kDa membrane lipoprotein of the syphilis spirochete, Treponema pallidum, and the outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi. These lipopeptides, which were synthesized without detectable endotoxin contamination, exhibit macrophage-stimulating activity that is not expressed by the corresponding non-acylated hexapeptides. The data indicate that synthetic lipopeptides based on spirochetal lipoproteins are appropriate substitutes for the intact lipoproteins in immunological studies.

Polyclonal antisera specific for the proenzyme form of each calpain.

Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes are exposed to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. We have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-terminal epitope of milli-calpain was used to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitope of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of mu-calpain in rat erythrocytes treated with ionomycin and calcium.

Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity.

The 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum was recently found to be a proteolipid. Similar two-dimensional electrophoretic mobilities and common hydrophobic properties displayed by the native (T. pallidum) and recombinant (Escherichia coli) 47-kDa antigens suggested that the recombinant antigen also possesses covalently bound lipid. Both intact E. coli and E. coli minicells acylated the 47-kDa antigen; immunoprecipitation with a monoclonal antibody specific for the 47-kDa immunogen supported the contention that the acylated product of E. coli corresponds to the cloned T. pallidum antigen. Triton X-114 phase partitioning was used to compare the relative hydrophobicities of 47-kDa molecules synthesized by in vitro and in vivo protein translation systems. The products synthesized by T. pallidum, intact E. coli, or E. coli minicells were hydrophobic, while the protein synthesized in an E. coli cell-free translation system was hydrophilic. Processing experiments with E. coli suggested that the primary gene translation product of the protein is not synthesized in a precursor form, unlike other bacterial proteolipids. These results indicate that the hydrophobicity of the 47-kDa integral membrane protein is conferred substantially by the covalently attached lipid(s). The biochemical similarities between the native and recombinant 47-kDa proteolipids will provide a foundation for future investigations into the structure and immunogenicity of this integral membrane protein of T. pallidum.

Improved fibrin plate method for fibrinolytic activity measurements: use of bentonite precipitation and agar solidification.

A modification of the fibrin plate method to assure sterility and fibrin stability is described. Bovine fibrinogen is precipitated with bentonite to remove the plasminogen and agar is added to assure stability. Storage of the plates up to 7-10 days has resulted in no bacterial growth. This rapid, sensitive method is useful in the isolation of plasminogen or its activators by chromatographic separation.