Characterization of secondary structure of neocarzinostatin apoprotein.

Characteristics of the secondary structure of neocarzinostatin apoprotein (apo-NCS) were examined by various means. Gaussian analysis of the Fourier-transform infrared (FT-IR) curve and curve-fitting of the circular dichroism (CD) spectrum for apo-NCS revealed that this peptide was abundant in beta-structures. In the presence of sodium dodecyl sulfate (SDS), the CD bands of NCS originating from phenylalanyl, tyrosyl, and cystinyl residues decreased, indicating a conformational change around the chromophore (CNS-chr). On the other hand, apo-NCS, in the SDS system, showed no change of these bands. We showed that the major parts of the protein moiety consist of beta-structures by measurements of the FT-IR and CD spectra of apo-NCS and a prediction of the secondary structure based on the amino acid sequence of the peptide. It seems that properties of the protein may be important to the hydrophobic interaction between NCS-chr and apo-NCS.

An antitumor polypeptide antibiotic neocarzinostatin: the mode of apo-protein--chromophore interaction.

The mode of neocarzinostatin-chromophore (NCS-chr)-apo-neocarzinostatin (apo-NCS) interaction in neocarzinostatin (NCS) complex has been described. The NCS-chr release from the NCS complex in the presence of various reagents, which destroy the high-order structure of the protein under various pH conditions, was examined. We found that (i) sodium dodecylsulfate, Nonidet P 40, 8 M urea, and 2-propanol did release NCS-chr from NCS, (ii) no NCS-chr release is detected below pH 7, but it is enhanced at high pH and (iii) beta-naphthol as a model of naphthalenecarboxylic acid derivative and D-galactosamine as a model of N-methylfucosamine of NCS-chr did release NCS-chr from NCS. These observations indicate that the binding of NCS-chr to apo-NCS may be due to not only ionic interaction between the acidic side chain of apo-NCS and the basic center of an aminosugar moiety of NCS-chr but also hydrophobic interaction between the hydrophobic amino acids of apo-NCS and hydrophobic moieties of NCS-chr. Apo-NCS is a very hydrophilic protein, since it has an high hydrophilic amino acid content. So, local hydrophobicity, local hydrophilicity and secondary structure of apo-NCS were predicted. Hydrophobic residues of apo-NCS predominantly located in beta-sheet structures near the carboxyl-terminus. These predictions are in good agreement with the results suggesting that NCS-chr bound carboxyl-terminal-43-peptide of apo-NCS in our previous result.

Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease-resistant sites in defined-sequence DNA.

A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.

Isolation and fast purification of neocarzinostatin by FPLC-ion exchange chromatography.

Neocarzinostatin, a highly toxic antitumor protein containing an essential nonprotein chromophore, can be isolated and purified from culture filtrates of Streptomyces carcinostaticus. Usually a lengthy procedure of up to 60 h is necessary for the isolation, including several chromatographic steps partly under conditions which favour inactivation of the drug by release of chromophore. We describe a new method yielding practically clinical grade Neocarzinostatin from crude extracts in 20 min. This very fast and reproducible method was made possible by using a Mono Q anion exchange column filled with monodisperse gel material which has been recently developed.

Enzyme immunoassay of neocarzinostatin using beta-galactosidase as label.

A novel convenient procedure was introduced for enzyme labelling of neocarzinostatin (NCS) with beta-D-galactosidase using a new hetero-bifunctional reagent N-(gamma-maleimidobutyryloxy) succinimide (GMBS). With the enzyme labelled NCS and a rabbit anti-NCS serum which was elicited in a rabbit immunized with NCS in complete Freund's adjuvant, a highly sensitive enzyme immunoassay which can quantify a femto mol order NCS was developed. Accuracy and precision of the assay as well as a comparison with the immunodiffussion method were studied with satisfactory results. This enzyme immunoassay of NCS was applicable to monitoring serum NCS levels of patients in clinical treatment of the antitumor agent.

Urea treatment and pronase digestion of antitumor protein antibiotics, auromomycin and neocarzinostatin.

Low molecular weight substances were separated from antitumor protein antibiotics, auromomycin and neocarzinostatin, by Sephadex G50 column chromatography, after denaturation with 8 M urea. The low molecular weight fraction of auromomycin, but not the protein fraction, showed antimicrobial and DNA-cleaving activities. More than 90% of the auromomycin and neocarzinostatin proteins were digested with a high concentration of pronase E. The digested samples of both antibiotics exhibited the same degree of activities as the original drugs in the inhibition of growth and DNA synthesis of mouse lymphoblastoma L5178Y cells and in causing strand scission of isolated PM2 phage DNA. The low molecular weight chromophores were recovered on Sephadex G50 column from the pronase-digested antibiotics. The results suggest that the in vitro biological activity of auromomycin and neocarzinostatin are principally attributed to the non-protein compounds of low molecular weight.

The promoting effect of amphotericin B on the incorporation of neocarzinostatin into human gastric cancer tissue.

AmB/NCS combination therapy against cancer was evaluated. Six gastric cancer patients were treated in this manner, and 7 gastric cancer patients treated with NCS alone to serve as the control. AmB sirup was administered orally for 4 days before surgical operation. NCS was given intraveously at the onset of gastric surgery. Lesion tissues and healthy tissues were collected from each patient and the NCS titers were measured by bioassay. It was shown in the majority of the gastric cancer cases that the NCS levels in the lesion tissues were substantially higher than in the surrounding normal tissues, whereas in the gastric cancer patients who received NCS alone, no significant differences were found between the tissues.

A lipophilic derivative of neocarzinostatin. A polymer conjugation of an antitumor protein antibiotic.

A lipophilic derivative of neocarzinostatin (NCS), an antitumor antibiotic, was prepared by reaction with a synthetic water-soluble polymer, [(styrene)1 approximately 3-(maleic acid 4 approximately 7/anhydride 1)]. The reaction was carried out at pH 8.6 for 3 h and aimed at modifying the two nonessential amino groups (alpha-amino of Ala-1, epsilon-amino of Lys-20). The NCS-polystyrene (SMANCS) was purified on a column of Sephadex G-100 in 0.05 M ammonium bicarbonate and the main product was obtained as a single peak. The elemental analysis showed an increased C and a decreased N content. U.v. and i.r. absorption spectra for SMANCS showed the presence of styrene. SDS-acrylamide gel electrophoresis at pH 8.5 and the decreased N content suggested a molecular weight of about 25 000, indicating the numbers of polymers conjugated to be about six units, two of which were found attached to the two amino groups. SMANCS was soluble in organic solvents, in contrast to NCS, and in water. SMANCS exhibited increased chemical and biological stability and appeared to possess similar in vitro biological activity.

Radioimmunoassays for methotrexate, citrovorum factor, neocarzinostatin, and actinomycin D.

Antisera specific for methotrexate, citrovorum factor, actinomycin D, and neocarzinostatin have been elicited in rabbits by immunization with appropriate antigens. The antibodies have been used together with radiolabeled marker haptens to produce sensitive and specific radioimmunoassays for each of these drugs.

A facile method of purification of neocarzinostatin, an antitumor protein.

A three-step column chromatographic method for the purification of neocarzinostatin (NCS) from a crude preparation was described. The purified material was homogeneous by acrylamide gel electrophoresis, isoelectric focusing, amino terminal analysis, and immunologic criteria. Purified NCS was 40 times as active in the inhibition of growth of Sarcina lutea and twice as active against CCRF-CEM human leukemia cells in vitro as was the starting material. When assayed against P388 and L1210 mouse leukemias in vivo, the purified material showed a median increase in life-span of 119 and 72%, respectively.

Radioimmunoassay of neocarzinostatin, an antitumor protein.

Antibodies directed toward the antitumor protein neocarzinostatin (NCS) have been produced in a rabbit by immunization with a highly purified NCS preparation. The antiserum was monospecific and reversed the antibacterial activity of NCS against Sarcina lutea. It cross-reacted with chemically modified derivatives of NCS and mitomalcin but failed to cross-react with macromomycin. A radioimmunoassay procedure has been developed utilizing the antiserum and a biologically active 125I-labeled derivative of NCS. The lower limit of detection by this radioimmunoassay, which involves a double antibody technique for the separation of antibody-bound and free antigen, was 1 X 10(-13) mole. The sensitivity of the assay is such that serum levels of NCS can be determined accurately after administration of the drug to rats at a single dose of 2 mg/kg. Since NCS is now undergoing clinical trial, the radioimmunoassay of the drug will be a valuable tool in clinical pharmacological studies.