Search

Chicken secretin has been isolated and its structure determined. It is composed of 27 amino acid residues, and has an amidated C terminus. The amino acid sequence is His-Ser-Asp-Gly-Leu-Phe-Thr-Ser-Glu-Tyr-Ser-Lys-Met-Arg-Gly-Asn-Ala-Gln-Val-Gln-Lys-Phe-Ile-Gln-Asn-Leu-Met-NH2. The structure shows distinct similarities to that of porcine secretin, amino acid identities occur at 14 positions (residues 1 – 4, 6 – 9, 11, 14, 17, 20, 24 and 26) but considerable differences are also present among the remaining 13 positions. Chicken secretin further shows a clear structural similarity to the vasoactive intestinal peptide (37% identical positions), the gastric inhibitory peptide (30% identical positions) and to glucagon (52 % identical positions), suggesting wide evolutionary and functional relationships. [ABSTRACT FROM AUTHOR]

The complete assignment of the aromatic proton nuclear magnetic resonances of the three tyrosine residues in hen lysozyme is reported. These assignments were made using double resonance techniques, specific chemical modifications of one residue (Tyr-23), and by interpretation of the effects of paramagnetic lanthanide ions. Some aspects of the behaviour of the tyrosine residues are reported, including pK values, reactivity towards modifying agents and conformational mobility. [ABSTRACT FROM AUTHOR]

The identification and complete assignment of the C-2 and N-1 proton nuclear magnetic resonances (NMR) of the six tryptophan residues of hen lysozyme are reported. Identification of the resonances required a detailed examination of the spectra of the protein in H2O and in ²H2O, and involved the application of spin-echo and Carr-Purcell-Meiboom-Gill pulse sequences. Assignment was achieved by observing the effects on the NMR spectra of performing specific chemical modifications, of binding paramagnetic species (lanthanide ions and spin labels), of binding inhibitors and protons and of carrying out solvent exchange experiments. The problems involved in completion of assignment are fully discussed. In the course of performing experiments to make assignments, several interesting aspects of the behaviour of the tryptophan residues in the protein structure were observed and are discussed. [ABSTRACT FROM AUTHOR]

[3H]Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with [3H]oestradiol during a 24-h incubation at 20 °C, 2 h at 30 °C or 45 min at 37 °C. Both ‘soluble’ and ‘insoluble’ nuclear receptors were stable for at least 6 h at 30 °C and 37 °C but a proportion (approx. 30 %) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 °C. The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10 × 1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3–6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73–77%) of their oestrogen receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82 %) of receptor sites were located in the nucleus. A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 μg oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 μg oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 μg oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h. [ABSTRACT FROM AUTHOR]

Chicken egg yolk contains an adenosine deaminase that was investigated after purifying about 500 times. It has a pH optimum at 6.5, a Km of 6,6 × 10-5 mol/l and an approximate molecular weight of 14000; higher molecular forms could not be detected. It was compared with the adenosine deaminases of chicken liver and blood plasma. From this comparison it is evident that this protein has undergone certain changes during the successive events leading to its final structure (secretion by the liver, transport through blood plasma to the oocytes and development of the egg): a common subunit with an approximate molecular weight of 15 000 may be the basis of the physiological diversifications. Substrate specificity of the purified extracts extends to cytidine and guanosine also, although certain observations point to different enzymes being involved. Deoxyadenosine is also deaminated. Cu2+, Zn2+ and Pb2+ are inhibiting and free - SH seems essential for activity. [ABSTRACT FROM AUTHOR]

In order to determine the sequence of histone F3 by automatic Edman degradation, this protein was fragmented into a series of smaller fragments. Specific chemical cleavages rather than enzymatic degradations were applied in such a way as to yield a limited number of larger fragments. In the first step the histone F3 dimer was cleaved at the two methionine residues with cyanogen bromide. The three fragments were separated and purified by gel filtration. Their respective sizes were 90, 30 and 15 amino-acid residues. The two larger fragments were both cleaved with N-bromosuccinimide at tyrosine residues. They in turn yielded three and two fragments, respectively, which could be readily purified by gel filtration and carboxymethyl-cellulose ion-exchange chromatography. One of the larger fragments obtained by cleaving the 90 residue fragment at its tyrosine residues was further degraded by dilute acid into another three fragments and two free aspartic acid residues which also could be purified by gel filtration. The position of all the 10 fragments in the protein molecule became apparent by comparing the N- and C-terminal amino-acid residues of the starting material and the cleaved products after each cleavage. The simplicity of the peptide mixture after each cleavage resulted in an easy separation of the fragments. [ABSTRACT FROM AUTHOR]

Antisera were prepared by immunizing a rabbit with procollagen synthesized and secreted by cells from chick embryo tendons. Specific antibodies were then purified from the antisera by using an immunoadsorbent which contained the isolated NH2-terminal extensions of procollagen. The purified antibodies were shown to react specifically with intact procollagen as well as with procollagen in which the interchain disulfide bonds were ruptured by reduction under non-denaturing conditions. The antibodies also reacted with the pro-αl chain but not the pro-α2 chain isolated from the procollagen. There was no reaction after the intrachain bonds in the pro-α chains of the procollagen were reduced under denaturing conditions and alkylated. In the course of characterizing the antibodies it was shown that the NH2-terminal extensions on the two pro-αl chains and the one pro-α2 chain of type I procollagen are non-identical. Also, it was shown that the serum of chick embryos contains aa antigen which reacts with the specific antibodies to procollagen. [ABSTRACT FROM AUTHOR]

Neurotrophin 3 (NT-3) had specific high-affinity receptors (HNT-3R) in the developing chick retina at all ages between embryonic day (E) 4 and E14. The affinity of HNT-3R for [sup 125]I-NT-3 did not change with the developmental state. A dissociation constant (k[sub d] of 13 pM was obtained. However, the amount of HNT-3R appeared to be developmentally regulated; the number of receptors/cell increased from E4 up to E6-7 (coinciding with the onset of neuronal differentiation), then decreased until E9 and increased again by E12, when all retinal cells were differentiated. Kinetic and cross-linking experiments showed that HNT-3R from two prototypical development ages, E7and E14, were different, E7 and E14 HNT-3R could be distinguished from each other on the basis of different inhibition patterns of [sup 125]I-NT-3 binding in the presence of nerve growth factor or brain-derived neurotrophic factor. Chemical cross-linking of increasing concentrations of [sup 125]I-NT-3 to its receptors showed (a) one 100-kDa band corresponding to neurotrophin low-affinity receptors in both E7 and E14 cells; (b) one 130-kDa band also present in both E7 and E14 cells. Densitometric measurements showed that this 130-kDa band behaved as HNT-3R in E14 cells (k[sub d]≈10 pM) but not in E7 cells (k[sub d]≥0.2 nM). Furthermore, the 130-kDa band in both E7 and E14 retinal cells displayed a trk-like immunoreactivity. Our data show that, in neurons, one particular neurotrophin may induce different actions mediated through distinct and specific receptors. [ABSTRACT FROM AUTHOR]

Each chick liver glutathione S-tranaferase CL 3 subunit contains three residues: His 142, Hist 158 and His 228. CL 3 –3 can be inactivated by treating with diethylpyrocarbonate. The inaction process is pH dependent and the pK of the modified residue is 6.4. The second-order inhibition rate constant is 741 M &sup -1; min &sup -1; at pH 7.0. Based on difference-spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. However, hydroxylamine treatment of the diethlpyrocarbonate-modified enzyme only partially restored the activity (30—50%) of Cl 3–3 tryptic mapping and amino acid sequence analysis. His 228 and Lys 14 have been identified as the modified residues. Mutants with histidine to serine replacement (H 142 S and H 158 S) or C-terminal histidine deletion (des-H 228) were constructed and over-expressed in Spodoptera frugiperda cells using a baculovirus system. The mutants are enzymically active. Furthermore, the des-H 228 mutant can be inactivated by diethylprocarbonate. These results support the conclusion that histidine are not involved in the enzyme mechanism of CL 3–3. [ABSTRACT FROM AUTHOR]

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na+-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]0 = 100 mM; [Na+] i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na+-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [³H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na+-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecaldiferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels. [ABSTRACT FROM AUTHOR]

We isolated a cDNA clone for a new isoform of chicken smooth muscle myosin regulatory light chain (MRLC) from a cDNA library of embryonic chicken gizzard. The deduced amino acid sequence was different in 10 amino acid residues from the previously reported polypeptide sequences of chicken gizzard MRLC. The hi vitro transcription/translation product from the cDNA comigrated with a minor isoform of chicken gizzard MRLC (L20-B) in a two-dimensional gel electrophoresis. This isoform was detected only in the embryonic gizzard and was slightly more acidic than the predominant isoform (L20-A). The partial polypeptide sequence of L20-A was confirmed to be identical to the previously reported MRLC sequence. Nevertheless, Northern blot analysis showed that L20-B-related mRNAs were present in both the embryonic and adult gizzard. Non-denaturing pyrophosphate polyacrylamide gel electrophoresis showed that the in vitro transcription/translation product could be associated with native myosin when mixed and coprecipitated in a low-ionic-strength buffer with adult chicken gizzard myosin. Moreover, the coprecipitated translation product was phosphorylated in vitro by chicken gizzard myosin light chain kinase apparently more rapidly than L20-A on the native myosin heavy chain. From these findings, we concluded that at least two isoforms of smooth muscle MRLC exist in chicken gizzard and that their expression may be regulated translationally depending on the developmental stage. [ABSTRACT FROM AUTHOR]

High-molecular-mass protein was isolated from chicken heart muscle. The apparent molecular mass of a single polypeptide chain is similar to that of chicken gizzard filamin: 250–270 kDa. The protein interacts with antibodies against chicken gizzard filamin and induces F-actin gelation in a concentration-dependent manner. Immunofluorescent staining of cardiomyocytes and chicken heart sections with antifilamin antibody demonstrates two types of filamin localization: (a) filamin was located on the sarcomere border in the periphery of the Z-disk; (b) filamin was found in intercalated disks between cardiomyocytes. [ABSTRACT FROM AUTHOR]

5′-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5′-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E. M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J. P. (1984) Biochem. J. 221, 369–377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5′-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5′-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5′-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver. [ABSTRACT FROM AUTHOR]

As part of a .study of the transfer of proteins and lipids from avian blood to egg yolk, some properties of lipoproteins from the blood of laying hens were compared with those of the low-density lipoprotein (YLP) from egg yolk of the same hens. Although it is known from previous work that particles of the very-low-density lipoprotein (VLDL) of blood arc the most likely precursors of YLP. their apoprotein patterns are different, according to electrophoresis and chromatography, with only one protein in common. YLP has the more complicated pattern which does not, however, include apoprotein B(ApoB) the main apoprotein of VLDL. It is suggested that during the transfer of VLDL to yolk. ApoB is cleaved to give smaller yolk apoproteins, especially apovitellenins IV and VI. Some evidence for this suggestion from the similarity of protein digests is presented. [ABSTRACT FROM AUTHOR]

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphoshorylated and monophosphorylated Src-CD (50 min-1), but the apparent Km values differ significantly (approximately 3 μM and 10 μM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for hi vivo activity. The apparent Km values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding. [ABSTRACT FROM AUTHOR]

Transcriptional control elements of chicken ribosomal protein L37a gene were characterized in terms of their activities to promote transcription and their protein binding activities. The region -120 to +168 was necessary for the maximal expression of the promoter-less CAT gene in a transfected chicken cell line. Using the DNase I protection assay, we identified nine protein binding regions distributed in a wide range of -122 to +195. The sequences of most of the elements are conserved among many vertebrate ribosomal protein genes at similar positions of the promoters. These common control elements and their binding proteins may coordinate the expression of ribosomal protein genes. [ABSTRACT FROM AUTHOR]

Mitochondrially bound dihydroorotate debydrogenase catalyses the fourth sequential step in the denovo synthesis of uridine monophosphate, 312-bp and 983-bp regions of the human dihydroorotate dehydrogenase sequence (1496 bp) were amplified by the polymerase chain reaction, and subcloned into the expression vector pQE 32, The identity of she PCR products was verified by dideoxynucleotide sequencing, Transformation of Eschereichia coli strain M 15 resulted in expression of 13-kDa and 36-kDa proteins with an affinity tag consisting of six consecutive histidine residues: these proteins could be purified by solubilisation in 8 M urea and by chromatography on a Ni2+ -chelating resin. In immunoblotting analyses, the fusion proteins were recognised by polyclonal avian and mammalian anti-peptide immunoglobulins. These were generated against synthetic peptides corresponding to two amino acid sequences deduced from human and rat cDMA of dihydroorotate dehydrogenase. The peptides were synthesized as multiple copies on a branching lysyl matrix. Rabbits and laying hens were immunized with these peptides without conjugation to a carrier protein. Comparison of the anti-peptide immunogobulins produced from egg yolk and rabbit serum demonstrated that avian anti-(dihydroorotate dehydrogenase) immunoglobulins may be considered a superior alternative to the mammalian equivalent: antibodies from both sources were applicable for all immunochemical purposes. Here, these antibodies were applied for identification of a 44-kDa protein from rat liver mitochondria, which was correlated with dihydroorotate dehydrogenase activity. [ABSTRACT FROM AUTHOR]

The crystal structure of chicken transthyretin has been solved at 290-pm resolution by molecular- replacement techniques. Transthyretin is the protein component of the amyloid fibrils found in patients suffering from either Familial amyloidotic polyneuropathy or senile systemic amyloidosis. Familial amyloidotic polyneuropathy is an autosomal dominant hereditary type at amyloidosis which involves transthyretin with either one or two amino acid substitutions. The three-dimensional structure of chicken transthyretin was determined in order to compare a non-arnyloidogenic, species-variant transthyretin with wild-type and mutant transthyretin molecules. Of the 31 chicken-to-human residue differences, 9 occur at positions which in human transthyretin give rise to amyloidogenie variants although none corresponds to the appropriate side-chain substitutions. The model of chicken transthyretin has been refined to an R- factor at 19.9%. The overall fold of the protein is that of an all-β protein. Compared with wild-type human transthyretin the avian transthyretin shows quite large differences in the region known to be involved in binding to retinal-binding protein, it has a much shorter helical component than the human protein and some of the monomer-monomer interactions are different. [ABSTRACT FROM AUTHOR]

A chicken B cell line DT40 and its syk-negative or lyn-negative mutants were used to investigate the roles of protein-tyrosine kinases in oxidant stress signaling. The data presented here for wild-type cells demonstrate that hydrogen peroxide stimulates p53/56lym-dependent tyrosine phosphorylation and activation of p72xyk, and induces a rapid and prolonged elevation of intracellular calcium, which consists of calcium release from intracellular stores and influx from the extracellular space. Hydrogen-peroxide-triggered calcium mobilization was impaired in both syk-negative and lyn-negative cells, which was mainly due to the loss of calcium release from intracellular stores. Further studies indicated that inositol trisphosphate production was also abolished in both syk-negative and lyn-negative cells, which is consistent with the loss of calcium release. Taken together, these observations suggest that the defect of p72xyk or p53/56lyn was responsible for the abnormality of calcium mobilization in both lyn-negative and syk-negative cells, and that both p72xyk and p53/56lyn might regulate calcium mobilization through the phosphatidylinositol pathway in B cell oxidant stress signaling. [ABSTRACT FROM AUTHOR]

The dissociation by NaCl of the nucleoprotein of chicken erythrocytes (histone/DNA=1.2, acid protein /DNA=0.24) has been systematically studied. The proteins dissociated at different ionic strength were characterized by amino-acid analysis and disc electrophoresis. The lysine-rich histone fraction is dissociated first byμ=0.4-0.5, the erythrocyte-specific histone fraction a little later by μ=0.6-0.7. Above μ=0.7 there is no further selectivity of dissociation. Up to an ionic strength of μ=0.3 there is practically no liberation of protein, then to μ=0.7 such liberation is linear. Between μ=0.7 and μ=1.0 the curve representing the quantity of histone liberated as a function of the ionic strength presents sections of different slope, indicating that the dissociation of histones occurs by successive stages in this region of ionic strength. Between μ=1.0 and μ2.0 the slope of the curve decreases continuously; at μ=2.0 the slope becomes essentially zero, indicating that histone dissociation is complete. A method for the preparation of the lysine-rich fraction and of the erythrocyte-specific fraction is proposed. A large percentage of nucleoprotein is precipitated by salt at ionic strength up to μ=0.6. At higher strength the nucleoprotein is redissolved progressively and the study of the residual complex is possible. The denaturation curves of the residual complexes obtained by dissociation of the nucleoprotein at these ionic strengths present two transitions, suggesting a heterogeneity of sites on the nucleoprotein molecule after dissociation by salt. [ABSTRACT FROM AUTHOR]