A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.

The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to proteins.

An improved method for the activation of polyethylene glycol with commercially available succinimidyl carbonate is described. The activated polyethylene glycol was coupled to proteins in high yield.

Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle.

We report here on the purification and characterization of a new 25-kDa inhibitor of actin polymerization from turkey gizzard smooth muscle. The protein was purified by chromatography on DEAE-cellulose and hydroxyapatite, as well as by affinity chromatography on an immobilized-antibody column. The purified polypeptide reduced the low-shear viscosity of actin, apparently due to its inhibitory effect on actin polymerization. We demonstrate that this protein is largely responsible for the apparent inhibitory activity previously reported to be associated with smooth muscle vinculin preparations. Three independent monoclonal antibodies prepared against the 25-kDa inhibitor of actin polymerization can effectively adsorb the inhibiting activity of actin polymerization from the crude vinculin preparation or inhibit it. We also show here that the 25-kDa inhibitor of actin polymerization tends to undergo dimerization when maintained in non-reducing buffers, concomitant with the loss of its inhibitory activity. Immunohistochemical labeling of frozen sections, as well as immunoblotting analyzes, indicated that the 25-kDa inhibitor of actin polymerization is particularly enriched in smooth muscle cells and that its distribution is apparently homogenous throughout the cytoplasm showing no apparent enrichment in the vinculin-rich dense plaques located along the endofacial surface of the plasma membrane.

Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology.

Avidin-biotin technology has been employed for the improved nonradioactive detection of glycoproteins on blots. Periodate oxidation of samples on blots converts the glycoprotein-based carbohydrate residues to the corresponding aldehydes. The latter undergo interaction with preformed complexes consisting of either avidin hydrazide or streptavidin hydrazide combined with biotinylated alkaline phosphatase. The sensitivity of the new assay exceeds the previously described enzyme hydrazide method by a factor of at least 10. The approach can be rendered selective for sialoglycoproteins, and approximately 12 sugar-containing bands could be observed in erythrocyte membrane preparations. Problems of nonspecific binding and high levels of background label were alleviated using a nonglycosylated basic protein (lysozyme) for quenching.

Improved method for preparing N-hydroxysuccinimide ester-containing polymers for affinity chromatography.

N,N,N',N'-Tetramethyl(succinimido) uronium tetrafluoroborate is proposed as a reagent of choice for the activation of carboxyl groups and formation of N-hydroxysuccinimide esters on polymers. Unlike conventional methods which generate unstable gels, the reaction is appropriate for hydroxy-containing resins like Sepharose, cellulose, and dextran. The yields of activation and subsequent coupling capacity for ligands and proteins are very high. The respective columns can be used for affinity chromatography and immobilization of proteins.

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies.

Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab' fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab'-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab' fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms.

Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum.

The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.