Osteotropic Peptide that differentiates functional domains of the skeleton

HPMA copolymer-d-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly discovered that D-Asp8 would favorably recognize resorption sites in skeletal tissues, while another bone-targeting moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8. In vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN. Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8 to the bone resorption sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the different levels of crystallinity of bone apatite at different functional domains of the skeleton.     

Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence

Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.     

Identification of a 5-HT4 receptor antagonist clinical candidate through side-chain modification

Replacement of the N-butyl side-chain of lead 5-HT4 receptor antagonist 2 with propanesulfonylpiperidinyl, morpholinyl, and piperazinyl groups led to higher affinity analogs 4-6. In vitro drug metabolism screens and cassette pharmacokinetic studies in the dog led to identification of the N-methylpiperazinyl analog (6b), which displayed pharmacokinetic, selectivity, and safety parameters sufficient for advancement to the clinic for the treatment of urinary incontinence.     

Regulation of hyaluronidase inhibition through its chemical modification

The modification of hyaluronidase by aldehydodextran regulates inhibition of the enzyme by heparin. A 70-90% modification of the surface amino groups of hyaluronidase results in sharp conformational changes and a substantial decrease of its inhibition by heparin, whereas hyaluronidase derivatives with a modification degree of 96-100% are practically uninhibited.     

Inhibition of phosphate transport across the human erythrocyte membrane by chemical modification of sulfhydryl groups.

Effects of sulfhydryl-reactive reagents on phosphate transport across human erythrocyte membranes were examined using 31P NMR. Phosphate transport was significantly inhibited in erythrocytes treated with sulfhydryl modifiers such as N-ethylmaleimide, diamide, and Cu2+/o-phenanthroline. Quantitation of sulfhydryl groups in band 3 showed that the inhibition is closely associated with the decrease of sulfhydryl groups. Data from erythrocytes treated with diamide or Cu2+/o-phenanthroline demonstrated that intermolecular cross-linking of band 3 by oxidation of a sulfhydryl group, perhaps Cys-201 or Cys-317, decreases the phosphate influx by about 10%. The inhibition was reversed by reduction using dithiothreitol. These results suggest that sulfhydryl groups in the cytoplasmic domain of band 3 may play an important role in the regulation of anion exchange across the membrane.     

Cytokeratin domains involved in heterotypic complex formation determined by in-vitro binding assays

Cytokeratins are constituent proteins of intermediate filaments (IFs) that form heterotypic tetrameric IF subunits containing two polypeptide chains of each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the basic (type II). To locate the molecular domains involved in the formation of these heterotypic complexes, we have developed a binding assay in which total cellular or cytoskeletal polypeptides, or proteolytically prepared cytokeratin fragments, are separated by one-, or two-dimensional gel electrophoresis, blot-transferred on to nitrocellulose paper and probed with radio-iodinated purified cytokeratin polypeptides or fragments thereof, using buffers of various ionic strengths with or without 4 M-urea. Using these polypeptides in the binding assay, specific heterotypic binding was observed between complementary cytokeratin polypeptides of the two subfamilies (but not with other IF proteins) and between the corresponding alpha-helical rod domain fragments. Both rod coils 1 and 2 of the type II cytokeratin 8 bound to the rod (coils 1 and 2) fragment of type I cytokeratins, and this binding occurred at both low and high ionic strengths. The results obtained indicate that: (1) the binding between cytokeratin polypeptides of the complementary type is stronger and more selective than interactions of cytokeratins with other IF and non-IF proteins; (2) both the head and the tail portions of the proteins are not required for heterotypic complex formation; (3) the complementarity information located in the alpha-helical portions of the rod domain, and in short sequences immediately flanking them, is sufficient to discriminate between the two types of cytokeratins and to secure the formation of heterotypic cytokeratin complexes; (4) both coils 1 and 2 of the rod can contribute to this association; and (5) the formation of the heterotypic cytokeratin complex is not critically dependent upon ionic interactions. Our results are further compatible with the concept that the heterotypic binding takes place between cytokeratin homodimer coiled-coils.     

I. Preparation of steroid-antigens through positions of the steroid not bearing functional groups

Dehydroepiandrosterone (DHA) and Dihydrotestosterone (dht) derivatives were prepared in order to obtain antibodies to these steroids. DHA and DHT were coupled to bovine serum albumin through positions which left the functional groups of the steroid free.An intercalated bond with a carboxylic function was linked to C-7 or C-15 of DHA and C-1 off DHT.     

Effect of chemical modification of amino groups by fluorescamine on partial reactions of photosynthesis.

4-Phenylspiro [furan-2(3H),1-phtalan]3,3'-dione (fluorescamine) was used to covalently modify amino groups of thylakoids. Subsequently its effect on parameters of energy transfer and phosphorylating activity was assessed. While electron transport, the extent of proton uptake, 515 nm change and 9-aminoacridine quench were relatively resistant to such treatment, the functions connected to coupling factor 1, namely ATP formation by acid/base transition, ATPase activity and photophosphorylation were affected much earlier. Photophosphorylation appears to be the most sensitive. The data are interpreted as indicating an involvement of free amino groups in energy transfer.     

The chemical modification of the essential groups of beta-N-acetyl-D-glucosaminidase from Turbo cornutus Solander

The chemical modification of beta-N-acetyl-D-glucosaminidase (EC3.2.1.30) from Turbo cornutus Solander has been first studied. The results demonstrate that the sulfhydryl group of cysteine residues and the hydroxyl group of serine residues are not essential to the enzyme's function. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 278 nm and the fluorescence intensity quenching at 335 nm, indicating that tryptophan is essential residue to the enzyme. The modification of amino group of lysine residue by formaldehyde and trinitrobenzenesulfonic acid also inactivates the enzyme completely. The results show that lysine and tryptophan are probably situated in the active site of the enzyme. The modification of the imidazole residue and carboxyl group leads to inactivate incompletely, indicating they are not the composing groups of the enzyme active center, and they are essential for maintaining the enzyme's conformation which is necessary for the catalytic activity of the enzyme.     

Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent.

Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.