Botulinum toxin type B: an overview of its biochemistry and preclinical pharmacology.

Produced by Clostridium botulinum, botulinum toxins are high molecular weight protein complexes consisting of the neurotoxin and additional nontoxic proteins that function to protect the toxin molecule. The neurotoxin acts to inhibit the release of acetylcholine at the neuromuscular junction, causing muscle paralysis. Purified toxin complexes have found a niche in the treatment of clinical disorders involving muscle hyperactivity. The different serotypes are structurally and functionally similar; however, specific differences in neuronal acceptor binding sites, intracellular enzymatic sites, and species sensitivities suggest that each serotype is its own unique pharmacologic entity. Recently, botulinum toxin type B has been developed as a liquid formulation to avoid the lyophilization (vacuum-drying) and reconstitution processes associated with decreasing the potency and stability of current type A toxin preparations. Biochemical tests were conducted to evaluate the quality of toxin in this formulation. In 3 consecutive manufacturing lots, the botulinum toxin type B complex was found to be highly purified, intact, uniform, and consistent from lot to lot. Also, it showed long-term stability at refrigerator and room temperatures (2 to 25 degrees C). Electrophysiologic studies in cynomolgus monkeys showed that botulinum toxin type B is effective in paralyzing injected muscle groups, with minimal spread to relatively distant noninjected muscles.

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity.

Cecropin A is a naturally occurring peptide with bactericidal activity against gram-negative and gram-positive bacteria. Production of large quantities of bactericidal peptides that are similar in structure and activity to cecropin A has been achieved by combining recombinant DNA techniques and techniques and chemical modification. Expression of the bactericidal peptide in Escherichia coli was accomplished through the formation of a fusion protein. The 5' end of the L-ribulokinase gene was fused to a single copy of a synthetic gene encoding cecropin A. A methionine codon was engineered between the two genes, and a methionylglycine extension was introduced at the C terminus of cecropin A. Cyanogen bromide treatment of the fusion protein yielded cecropin A with a C-terminal homoserine. The recombinant cecropin A with a homoserine at the C terminus did not kill most gram-positive bacteria tested. However, recombinant cecropin A with a chemically modified C terminus has antimicrobial activity similar to that of cecropin produced by cecropia pupae.

Affinity purification of tissue plasminogen activator using transition-state analogues.

The search for a simple affinity ligand to purify tissue plasminogen activator (tPA) was facilitated by a solid-phase synthesis approach. A large variety of tripeptide ligands containing argininal were synthesized on agarose gels containing a spacer with carboxy terminal. The immobilized ligands were easy to test with urokinase, and tPA. While a number of sequence combinations showed initial binding by tPA, only a few resulted in tight binding corresponding to a hemiacetal linkage with the active site serine. Hydrophobic residues, especially aromatics, flanking the N-side of argininal gave rise to ligands which were bound strongly by tPA. A gel containing D-Phe-D-Phe-Argal (an aldehyde derivative of arginine) was very effective in purifying tPA derived from cell culture media at small scale (milligrams) and at large (multi-grams).

Induction of collagenase production in U937 cells by phorbol ester and partial purification of the induced enzyme.

The U937 cell line is a monoblast-like cell line that can be induced to differentiate when treated with phorbol ester or a variety of other agents. Collagenase was detected in the media of U937 cell cultures after treatment with phorbol myristate acetate (PMA) at concentrations of 5 ng/mL or greater. In general, no collagenase was detected in the media of untreated cells. The induced collagenase cleaved native type I collagen into the 3/4 and 1/4-length fragments and showed the inhibition by ethylenediaminetetraacetic acid characteristic of the action of mammalian collagenases. Collagenase activity could be detected in the media of treated cells 12-18 h after the addition of PMA. Secretion of collagenase continued for 2-3 days after PMA addition. The production of collagenase by PMA-treated U937 cells was inhibited by actinomycin D and cycloheximide, suggesting that the induction of the enzyme is the result of de novo synthesis. The collagenase secreted by U937 cells induced with PMA has been purified 12-fold by using DEAE-Sephacel followed by wheat germ agglutinin-agarose chromatography. The apparent molecular mass of this U937 collagenase, determined by gel filtration chromatography on the partially purified enzyme, was 29-36 kilodaltons.

Use of lectin affinity chromatography for the purification of collagenase from human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNLs) store collagenase in an inactive form in secretory granules. The enzyme can be activated in vitro by limited proteolysis or by sulfhydryl-modifying agents such as N-ethylmaleimide (NEM). We have enriched NEM-activated collagenase 820-fold using granule isolation, gel filtration, and wheat germ agglutinin (WGA)-agarose chromatography. The use of WGA-agarose resulted in a 55-fold enrichment of collagenase in a single step with very little loss of activity. The chromatographic behavior of collagenase on other lectin matrices was explored and gave information about the type of complex asparagine-linked oligosaccharide found on collagenase isolated from PMNLs.

Contact site of histones 2A and 2B in chromatin and in solution.

Irradiation of isolated nuclei or of a complex of histones 2A (H2A) and 2B (H2B) with ultraviolet light produces a covalent cross-link between H2A and H2B. Sequence analysis of the peptides isolated from the H2A-H2B dimer formed in solution and in nuclei demonstrated that both dimers are produced through the covalent linkage of Tyr-40 of H2B and Pro-26 of H2A. Tyrosyl residues proximal to Tyr-40 did not produce a cross-link with H2A, thereby indicating that strict conformational parameters are required for production of the H2A-H2B cross-link. We conclude that the precise juxtaposition of Tyr-40 of H2B and Pro-26 of H2A in this region of the H2A/H2B contact site is not altered upon interaction of these histones with H3 and H4 (tetramer), DNA, or other chromosomal components during nucleosome assembly.

Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex.

The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. We also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly.

C-protein from rabbit soleus (red) muscle.

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.