Tissue-specific distribution of a peroxisomal 46-kDa protein related to the 58-kDa protein (sterol carrier protein x; sterol carrier protein 2/3-oxoacyl-CoA thiolase).

The complete sequence of the nonspecific lipid-transfer protein (nsL-TP; sterol carrier protein 2) including the presequence is present at the C-terminus (residues 405-547) of a 58-kDa protein. To be able to study this 58-kDa protein without the interference of nsL-TP, antibodies were raised against predicted epitope regions in the N-terminal part (peptide I, residues 23-43; peptide II, residues 130-149). Using these antibodies, rat tissues were analyzed by immunoblotting. In rat liver, in addition to the 58-kDa protein the antibody against peptide I (alpha-58K23) as well as the antibody against peptide II (alpha-58K130) detected a 46-kDa protein. This suggests that both peptide sequences are present in this 46-kDa protein. Both the 46- and the 58-kDa-proteins were abundantly present in liver and adrenals, but could also be detected in brain, kidney, heart, lung, testes, and ovary. This distribution was observed in tissues from both male and female rats. Immunogold labeling of cryosections of liver showed that alpha-58K23 labels the peroxisomes. From double-labeling experiments using alpha-nsL-TP and alpha-58K23 we conclude that the 46-kDa protein is peroxisomal. We propose that in the peroxisomes the protease that processes pre-nsL-TP also cleaves the 58-kDa protein giving rise to the 46-kDa protein and nsL-TP. In addition to the 58- and 46-kDa proteins, an immunoreactive 44-kDa protein was prominently present in rat heart and at low levels also in small intestine and brain. Immunogold labeling of cryosections of heart and Western blotting of purified mitochondria showed that the 44-kDa protein is localized in the mitochondria. The 44-kDa protein was shown to be identical to mitochondrial sarcomeric creatine kinase, which has a peptide segment of five amino acid residues in common with peptide I.

An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system.

An isoform of the phosphatidylinositol-transfer protein (PI-TP) was identified in the cytosol fraction of bovine brain. This protein, designated PI-TP beta, has an apparent molecular mass of 36 kDa and an isoelectric point of 5.4. The N-terminal amino acid sequence (21 residues) is 90% similar to that of bovine brain PI-TP, henceforth designated PI-TP alpha (molecular mass 35 kDa and pI 5.5). As observed for PI-TP alpha, PI-TP beta has a distinct preference for phosphatidylinositol over phosphatidylcholine. In addition, it expresses a high transfer activity towards sphingomyelin. PI-TP alpha lacks this activity completely. By indirect immunofluorescence we demonstrated that, in Swiss mouse 3T3 fibroblasts, PI-TP beta is preferentially associated with the Golgi system whereas PI-TP alpha is predominantly present in the cytoplasm and the nucleus. In cytosol-depleted HL60 cells, both PI-TP alpha and PI-TP beta were equally effective at reconstituting guanosine 5'-[gamma-thio]triphosphate-mediated phospholipase C beta activity.

Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli.

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.

Analysis of glycoforms present in two mouse IgG2a monoclonal antibody preparations.

Lectins have been used for the determination of the oligosaccharide structures expressed by two monoclonal IgG antibodies, MN12 and RIV6. Dot blot experiments revealed the presence of terminal Fuc alpha (1-->6)GlcNAc, Gal beta (1-->3)GalNAc, Gal beta (1-->4)GlcNAc, Man alpha (1-->6, 1-->3)Man, NeuAc alpha (2-->6)Gal and NeuAc alpha (2-->6)GalNAc on both monoclonal antibodies. MN12 was shown to contain a carbohydrate moiety within the Fc region only. RIV6 contained carbohydrate moieties within both the Fc and Fab regions. Additional O-glycosidic linked carbohydrate chains were detected within the Fc region of both monoclonal antibodies. High mannose structures were also detected on both Mabs.

Instability of a hybridoma cell line in a homogeneous continuous perfusion culture system.

In this study several analytical techniques were applied to obtain information about the stability of expression, the yield, and the integrity of a monoclonal antibody (Mab) produced by hybridoma cell line RIV6 in a homogeneous continuous perfusion culture system. The total antibody as well as the isotype-specific antibody contents decreased continuously during the course of cultivation, while the viable cell concentration remained constant. The origin of the discrepancy between the Mab contents observed by two enzyme-linked immuno sorbent assay (ELISA) systems during the steady state was due to fragmentation of the IgG molecule, either cytoplasmic or in the culture fluid, as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The IgG-secreting cells as well as the fraction of cells containing a high cytoplasmic IgG content decreased continuously during cultivation. The isoelectric focusing (IEF) pattern showed the appearance of two additional bands after five days of cultivation. This work indicates that cell line RIV6 is unstable in the culture system used, with respect to cell properties and product formation.