Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C.

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.

Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line.

We examined the role of phosphatidylcholine-specific phospholipase D (PC-PLD) in the IgE-dependent activation of protein kinase C (PKC) in RBL 2H3 cells (a model for mast-cell function). Cells were sensitized with mouse monoclonal anti-trinitrophenol (TNP) IgE (0.5 micrograms/ml) and were then triggered with an optimal concentration (10 ng/ml) of TNP-ovalbumin conjugate (TNP-OVA). This resulted in an immediate biphasic increase in the production of 1,2-diacylglycerol (DAG) and activation of PKC. The initial increase in DAG production reached a peak within 30 s, and the second phase reached a plateau within 5 min after stimulation. TNP-OVA-induced PC-PLD activation followed the initial increase in DAG formation in response to IgE-receptor cross-bridging, but coincided with the second peak. Phosphatidic acid (PA), derived from the PC-PLD pathway, is metabolized to DAG by the action of PA phosphohydrolase (PAPase). Propranolol (0.3 mM), which inhibits PAPase, blocked the IgE-dependent increase in DAG, activation of PKC, and subsequently degranulation. The PKC inhibitor staurosporine (0.1 microM) inhibited the second, but not first, peak of DAG accumulation, reversed PKC translocation after 10 min and inhibited subsequent mediator release. Taken together, these results demonstrate that PC-PLD does not initiate, but may play a latent role in, IgE-dependent DAG production, PKC activation and mediator release from RBL 2H3 cells.

Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D.

A phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) that specifically hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol-glycans (PI-Gs) has recently been purified from human and bovine sera. The primary structure of bovine PI-G PLD has now been determined and the functional activity of the enzyme has been studied. Expression of PI-G PLD complementary DNA in COS cells produced a protein that specifically hydrolyzed the inositol phosphate linkage of the PI-G anchor. Cotransfection of PI-G PLD with a PI-G-anchored protein resulted in the secretion of the PI-G-anchored protein. The results suggest that the expression of PI-G PLD may influence the expression and location of PI-G-anchored proteins.

Purification and characterization of glycosyl-phosphatidylinositol-specific phospholipase D.

We have developed a simple immunoaffinity chromatography procedure for the purification of a glycosyl-phosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) from bovine serum. The enzyme was initially purified by a procedure consisting of 9% polyethylene glycol precipitation, Q Sepharose anion-exchange chromatography, S-300 gel filtration, wheat germ lectin-Sepharose, hydroxylapatite agarose, zinc chelate matrix, Mono Q-high performance liquid chromatography (HPLC), and Superose 12 (gel filtration) HPLC. Using this purified material as immunogen, we generated a panel of monoclonal antibodies. A low affinity antibody was selected for the purification of catalytically active GPI-PLD from bovine serum by immunoaffinity chromatography, followed by wheat germ lectin-Sepharose and Mono Q-fast protein liquid chromatography. The latter method provides a simple purification procedure with an overall yield of 26%. The purified enzyme has an apparent molecular weight of about 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of about 5.6 by isoelectric focusing gel analysis. On Superose 12 HPLC, the material purified by the latter method elutes as a single peak with an apparent molecular weight of 200,000 as determined by protein standards. The enzyme activity is inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid or 1,10-phenanthroline. Phosphatidic acid is the only 3H-labeled product when [3H]myristate-labeled variant surface glycoprotein is hydrolyzed by the purified enzyme. Amino terminal sequence analysis of the intact 100-kDa protein reveals no strong homology to that of any other known protein. Twelve tryptic peptides derived from the intact protein have been subjected to amino acid sequence analysis. Two of them share sequence homology with each other and with the metal ion binding domains of members of the integrin family. Based upon these criteria, it appears that the purified enzyme is distinct from other phospholipases with specificity for inositol phospholipids.

Proton diffusion in the active site of triosephosphate isomerase.

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)