Peter M. Grob

INACTIVATION OF CELL SURFACE RECEPTORS BY PHEOPHORBIDE a, A GREEN PIGMENT ISOLATED FROM Psychotria acuminata

Abstract— The inhibition of cytokine and monoclonal antibody binding to cell surfaces caused by an extract of Psychotria acuminata, a medicinal plant used in the traditional medicine of the people of Belize (Central America), was attributed to the presence of pheophorbide a and pyropheophorbide a Since the binding of tumor necrosis factor‐alpha, interleukin‐8, complement factor 5a as well as epidermal growth factor to target cells was dramatically reduced, the inhibition was not receptor or cytokine specific. In addition, the respective binding of radiolabeled monoclonal antibodies CL203 and R15.7 to the cell surface antigens intracellular cell adhesion molecule‐1 and lymphocyte function‐associated antigen‐1 ß‐chain was decreased by pretreatment of cells with pheophorbide a as well. In all cases, the inhibition by pheophorbides was dependent on the simultaneous presence of light, indicating causative involvement of a photodynamic process. These observations are not unique to pheophorbides and can be extended to porphyrins as well as to other photodynamic agents. Cytotoxicity resulting from photodynamic therapy (PDT) has been documented by many studies. Our investigations suggest that the inactivation of cell surface receptors contributes not only to the antitumor effect of PDT but also to the systemic immunosuppression, a serious side effect of PDT.

High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus type-1

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: Preparation of heterodimeric enzyme devoid of unprocessed gene product

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.

[80] Separation and characterization of molecular components of human leukocyte interferon by concanavalin A-agarose affinity chromatography

Publisher Summary This chapter reveals that human leukocyte interferon (HLIF) is composed of several homologous proteins that are coded for by separate genes in the genome. HLIF can be resolved into two molecular weight components by sodium dodecyl sulfate (SDS)-disc gel electrophoresis and, in addition, has been shown to contain fibroblast-type interferon as a minor component. These three molecular components: (1) 21,000-dalton component (Le21), (2) 16,000-dalton component (Le16), and (3) a fibroblast- type minor component (“F” type) cannot be effectively resolved either by gel filtration or by SDS-disc gel electrophoresis. The chapter describes the efficient separation of these three molecular components, in a single step, by chromatography of HLIF on concanavalin Aagarose. NDV is routinely used as an interferon inducer and human serum is present throughout the production period. It is conceivable that the nature or extent of molecular heterogeneity will vary if these production conditions are changed.