ABSTRACT
Thai bitter gourd protein (MRK29) was isolated from Momordica charantia ripe fruit and seed. The purification was performed by ammonium sulfate fractionation and gel filtration chromatography. MRK29 possessed one isoelectric point of (pI) > or = 9, and the time of flight mass spectrum (TOFMS) indicated its molecular weight at 28.6 kD. The twenty amino acid sequence from the N-terminus was in the following order: 1Asp Val Asn Phe Arg Leu Ser Gly Ala 10Asp Pro Arg X Tyr Gly Met Phe Ile Glu 20Asp. MRK29 inhibited the HIV-1 reverse transcriptase with 50% IR at the concentration of 18 micrograms/ml. MRK29 was concentrated in the 30-60% salt precipitated fraction, at which the concentration of 0.175 microgram/ml exerted 82% reduction of viral core protein p24 expression in HIV-infected cells. MRK29 might have modulatory role on immune cells, because it increased 3-fold TNF activity.
Subject(s)
Anti-HIV Agents/isolation & purification , Cucurbitaceae/chemistry , Fruit/chemistry , N-Glycosyl Hydrolases , Plant Proteins/isolation & purification , Plants, Medicinal/chemistry , Reverse Transcriptase Inhibitors/isolation & purification , Seeds/chemistry , Amino Acid Sequence , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , HIV Core Protein p24/analysis , HIV Core Protein p24/immunology , Humans , Lymphocytes/immunology , Macrophages/immunology , Mass Spectrometry , Molecular Weight , Plant Proteins/chemistry , Plant Proteins/pharmacology , RNA-Directed DNA Polymerase/blood , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Ribosome Inactivating Proteins, Type 2 , Sequence Analysis, Protein , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Complete 1H-nmr data and unambiguous assignments of the 13C-nmr spectra of phyllanthin [1] and hypophyllanthin [2] were obtained through extensive nmr studies, including homonuclear COSY, homonuclear decoupling, APT, HETCOR, nOe difference, selective INEPT, and COLOC experiments. The absolute configuration of hypophyllanthin [2] was determined by cd. Neither of these lignans demonstrated significant cytotoxic activity when evaluated with a battery of cultured mammalian cells, but both were found to enhance the cytotoxic response mediated by vinblastine with multidrug-resistant KB cells. In addition, 1 was found to displace the binding of vinblastine with membrane vesicles derived from this cell line, suggesting an interaction with the P-glycoprotein.
