Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase.

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5' and 3' ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3'-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.

Monoclonal autoantibodies against mouse red blood cells: a family of structurally restricted molecules.

Cultured mouse peritoneal cells from unstimulated mice developed plaque-forming activity against isologous bromelain-treated erythrocytes. Several IgM monoclonal autoantibodies obtained by fusion of peritoneal cells from NZB or CBA origin with BALB/c myeloma cells were purified by affinity chromatography on trimethyl ammonium (TMA) column on the basis of their cross-reactivity with TMA, phosphorylcholine (PC) or choline haptens. Binding affinity for PC hapten was of the order of 10(3) M-1. Idiotypic studies with a polyclonal rabbit anti-idiotypic reagent revealed strong cross-reactions with all hybridoma autoantibodies thus far tested. In addition, the rabbit anti-idiotypic serum detected idiotypes or cross-reactive idiotypes in the sera of NZB and CBA as well as BALB/c mice. N-terminal amino acid sequence analyses of three hybridoma autoantibodies from NZB mice and one from CBA mice were carried out. The sequences of the first 32 residues of the four heavy chains showed that three were identical while one had one amino acid interchange; they belong to the VHIII-subgroup. The light chains were identical in the first 35 residues with the exception of a substitution at position 3 in two light chains and are members of the VK-9-subgroup. These results entirely support the idiotypic data. These monoclonal autoantibodies from NZB and CBA mice although isolated and eluted from PC-related haptens do not have any apparent structural nor idiotypic relationship to PC-specific antibodies. Idiotypic and V-region N-terminal sequence data suggest that these autoantibodies constitute a highly restricted family of molecules likely to be encoded by unique germ-line genes which may be expressed as such or as somatic variants in different mouse strains.