Determination of the sizes of the opioid receptors in their membrane environment by radiation inactivation

Opioids, like other drugs, are thought to initiate their effects by association with their specific receptors. However, very little is known about the opioid receptor as a molecular entity. The binding components have been solubilized in detergent and purified by different approaches, but the molecular size of soluble opioid receptor complexes reported by different groups varied from 23,000 to 750,000. In this study, the technique of radiation inactivation by gamma rays was used to investigate the apparent size of the opioid receptor in rat brain membranes under different conditions. The molecular sizes of opioid receptor complexes were estimated as 313,000 +/- 13,500 in the presence of [D-Ala2, D-Leu5] enkephalin, NaCl and Gpp (NH)p; as 165,000 +/- 8,500 in the presence of NaCl only, or of both NaCl and Gpp (NH)p; as 217,000 +/- 6,600 in the presence of Gpp (NH)p only; and as 286,000 +/- 60,900 in the presence of MgCl2 only. A simple model has been proposed to explain these different apparent target sizes of opioid receptors obtained under different conditions.     

Deletional Protein Engineering Based on Stable Fold

Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.     

Anti-coccidial activity of 2-picoline, 6-amino-4-nitro-, 1-oxide

An investigational drug (2-picoline, 6-amino-4-nitro-, 1-oxide) was evaluated to characterize the anti-coccidial spectrum of the compound. Two concentrations of the drug (125 and 250 ppm) were evaluated for bioactivity; weight gain, survival, dropping, and lesion scores were the response variables utilized to ascertain activity. The activities of the picoline derivative were compared with monensin, maduramicin, and a narasin/nicarbazin (1:1) combination. The investigational drug had significant activity against Eimeria tenella and Eimeria necatrix, and the 250-ppm level was significantly more active than 125 ppm. At 250 ppm, the E. tenella activity of the picoline derivative was comparable to both monensin (120 ppm) and the 50-ppm narasin/nicarbazin combination, significantly less effective than maduramicin (6 ppm), and significantly more efficacious than 30 ppm narasin/nicarbazin. At the same level (250 ppm), the picoline derivative had significantly less E. necatrix activity than monensin (120 ppm), maduramicin (6 ppm), and narasin/nicarbazin (50 ppm), and significantly greater activity than 30 ppm narasin/nicarbazin. At best, only extremely weak Eimeria acervulina, Eimeria brunetti, and Eimeria maxima activities were noted with the investigational drug; higher concentrations of the picoline derivative may achieve greater anti-coccidial activity against these species. The efficacy of narasin/nicarbazin compared favorably with monensin and maduramicin; the 50-ppm level of the combination appeared significantly more efficacious than 30-ppm.     

Immobilization of enzymes on Spheron: 2. β-Amylase — The development of a column reactor—separator

The titanium-chelation method has been used to immobilize β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) on to Spheron. On various grades of Spheron, protein coupling yields of 56–76% were obtained with barley and sweet-potato β-amylases. The specific enzymic activities of the immobilized enzymes fell in the range 3.7–7.6% of those of the soluble enzymes. The immobilized enzymes were more stable than the soluble, especially in the presence of l-cysteine and serum albumin. The presence of cysteine and serum albumin brought about increases in activity in the preparations, presumably by regenerating essential thiol groups in the enzyme which had been oxidized during the operations. Maltose could be separated from amylopectin and other large polysaccharides by chromatography on Spheron P100, and a system was developed in which maltose, produced by hydrolysis of amylopectin applied in pulses to a column of immobilized β-amylase, was separated from starting material and by-products on a second column of Spheron P100.     

Structural characterization of canine PYY

PYY was purified from canine colonic mucosa by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence, amino acid and mass spectral analyses of the purified peptide and its tryptic fragments were consistent with the structure: YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY-amide. Canine PYY(1-36) has the identical sequence as porcine and rat PYY but differs from human PYY at position 3, with Ala instead of Ile, and position 18, with Ser instead of Asn. A smaller form, PYY(3-36), was also purified and characterized. It may differ in its biological activity from the intact peptide and could act as a partial antagonist or agonist of PYY(1-36).     

Dynamic changes of horse serum T-globulin immunization with snake venoms, tetanus and diphtheria toxoids

In course of immunizing horses with snake venoms, tetanus and diphtheria toxoids, a new serum component, T-globulin, was formed and migrated between the beta- and gamma-globulins. The T-globulin content was parallel with the antibody titre after the middle course of immunization. There were many components in snake antivenin and T-globulin was composed of most of those components. The components of diphtheria T-globulin were the same as those of crude antitoxin and tetanus T-globulin except one precipitin.