Enzyme-bound early product of purified poly(ADP-ribose) polymerase.

Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-poly-acrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD⁺, ADP-ribose unit was found to be added to the terminal AMP-residue of the oligo(ADP-ribose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme.     

Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C-dextran conjugate.

Monoclonal antibody (mAb) A7, produced against human colon cancer, was conjugated with a polymeric prodrug of mitomycin C (MMC), the MMC-dextran conjugate with an anionic charge (MMCDan) and a molecular weight of 70,000. The amino groups were introduced into the MMCDan by reacting ethylenediamine with the carboxyl group in the spacer arm of the dextran bridge by a carbodiimide-catalyzed reaction. The coupling to mAb A7 was performed using SPDP. A 15 M excess of ethylenediamine produced an optimal MMCDan with amino groups, which resulted in a homogenous conjugate (A7-MMCD) with minimal formation of high-molecular-weight aggregates in about a 30% yield of both IgG and MMC. The molar binding ratio of IgG:dextran:MMC in A7-MMCD was estimated to be 1:1.2:40. A7-MMCD, having MMC prodrug properties, released active MMC with a half-life of 29.1 h and had an almost neutral electric charge under physiological conditions. A competitive binding assay using 125I-labeled A7 revealed that the A7-MMCD almost fully retained its antibody-binding activity. The cytotoxicity of A7-MMCD was assayed by determining the degree of inhibition of [3H]-thymidine in corporation in two different ways using the human colon cancer cell line SW1116. A 48-h continuous exposure test revealed that the pharmacological activity of MMC in A7-MMCD was completely preserved. In addition, A7-MMCD exhibited about a 14-fold greater cytotoxicity than MMCDan when the IC50 values determined using a 2-h pretreatment exposure system were compared. These results suggest that A7-MMCD could be useful in immunotargeting chemotherapy for colorectal cancer.     

Stimulation of bull seminal Ca2+, Mg2+-dependent endonuclease by various DNA-binding proteins

We have measured ΔA transient absorption spectra in the Soret region and kinetics of photodissociation of oxymyoglobin (MbO₂) solutions following excitation by pulses of duration 350 fsec and 10 μJ energy at 307 nm. We observed an instantaneous bleaching of the absorbance at 414 nm and the appearance of a broad, red-shifted absorption band in the 438–470 nm region with a time constant of 250 fsec indicative of the formation of a short-lived deliganded Mb^★ species which relaxes to the stable Mb with a constant of 3.5 psec. Following this early relaxation, changes in absorption kinetics indicate also a geminate recombination process of constant τ = 100 psec. These data demonstrate that the well established low quantum yield (φ = 0.03) of photodissociation in MbO₂ is related both to the relaxation of an excited Mb^★ state and to a fast geminate recombination process.