Stability of (2'-5')oligoriboadenylates in various sera

Avian and mammalian sera were found to contain an enzyme activity degrading 2-5A oligonucleotides. The most extensive degradation of the A2' p5' A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56 degrees C for 30 min. The 5-mU3' p5' A has also been degraded in chicken serum.     

Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate

Phosphoenolpyruvate carboxykinase from chicken liver mitochondria and rat liver cytosol catalyzes the phosphorylation of alpha-substituted carboxylic acids such as glycolate, thioglycolate, and DL-beta-chlorolactate in reactions with absolute requirements for divalent cation activators. 31P NMR analysis of the reaction products indicates that phosphorylation occurs at the alpha-position to generate the corresponding O- or S-bridged phosphate monoesters. In addition, the enzymes catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyzes the bicarbonate-dependent phosphorylation of fluoride ion. The kappa cat values for these substrates are 20-1000-fold slower than the kappa cat for oxaloacetate. Pyruvate and beta-hydroxypyruvate are not phosphorylated, since the enzyme does not catalyze the enolization of these compounds. Oxalate, a structural analogue of the enolate of pyruvate, is a competitive inhibitor of phosphoenolpyruvate carboxykinase (Ki of 5 microM) in the direction of phosphoenolpyruvate formation. Oxalate is also an inhibitor of the chicken liver enzyme in the direction of oxaloacetate formation and in the decarboxylation of oxaloacetate. The chicken liver enzyme is inhibited by beta-sulfopyruvate, an isoelectronic analogue of oxaloacetate. The extensive homologies between the reactions catalyzed by phosphoenolpyruvate carboxykinase and pyruvate kinase suggest that the divalent cation activators in these reactions may have similar functions. The substrate specificity indicates that phosphoenolpyruvate carboxykinase decarboxylates oxaloacetate to form the enolate of pyruvate which is then phosphorylated by MgGTP on the enzyme.     

gamma-Glutamyltranspeptidase and dipeptidyl peptidase IV activity in the serum of normal and hepatoma-bearing chickens and in the plasma membranes from liver and hepatoma Mc-29.

Investigations on the activity of gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) in the serum of healthy chickens and those bearing hepatoma Mc-29, and in liver and hepatoma plasma membranes were carried out. There was no difference in the serum enzyme activities of control and tumor-bearing chickens but the activity of GGT was twice higher and that of DPP IV 20 times lower in hepatoma plasma membranes than in chicken liver plasma membranes. Using thin-layer analytical isoelectric focusing in agarose gels it was established that the pI range of GGT from host serum and hepatoma plasma membranes was shifted to more acidic values. This could be interpreted as a specific feature for this enzyme considered as a tumor marker.     

Comparative study of the proteolytic products of fibronectins in the serum of adult and embryonic animals formed under the effects of proteinases of the spleen

The effects of two spleen proteinases, cathepsin D and thiol proteinase I active in neutral media--on the structural properties of fibronectins from blood plasma on adult animals and their embryos were investigated. Proteinase I and cathepsin D caused rapid fragmentation of all fibronectins under study. Fibronectin from calf embryonic serum was more sensitive to proteinase I than that from adult animal serum. The molecular weight and the correlation between the proteolytic products formed under the influence of each enzyme, on the embryonic and "adult" fibronectins, are very similar but not identical. Similar results were obtained in experiments with proteolytic products of chicken serum and embryo fibronectins. Fragmentation of embryonic fibronectin occurs more rapidly than that of chicken fibronectin; the fibronectin proteolytic products differ both qualitatively and quantitatively. However, the determination of structural differences between these fibronectins is considerably hampered by the presence of protein contaminations in chicken fibronectin preparations.     

Pyruvate kinase and creatine phosphokinase during development of the chicken with muscular dystrophy

Pyruvate kinase and creatine phosphokinase activities in breast muscle extracts and in serum, and protein content of the muscle extracts were determined during the first eight weeks of development of control and dystrophic chickens. In the dystrophic chicken serum enzyme levels were significantly greater than, and muscle protein content and enzyme activities on a gram wet weight basis were significantly less than control values, by the second week after hatching and thereafter. For both muscle and serum the relative differences between control and dystrophic groups was greater for pyruvate kinase than crearine phosphokinase. On a specific activity basis only pyruvate kinase levels in dystrophic muscle were significantly less than control values in 2–8-week-old chickens.     

Galactosyltransferase: multiple forms in serum of normal and hepatoma Mc-29 bearing chickens and from liver and hepatoma microsomal and plasma membrane preparations

The multiple forms of galactosyltransferase in chicken serum and in microsomal and plasma membrane preparations from liver and viral induced hepatoma Mc-29 have been studied by isoelectric focussing. An elevation of the hepatoma plasma membrane enzyme activity was described and in the pattern of the multiple forms of the enzyme two forms were found (pI-5.34 and 8.22) which were similar to those described in the serum of hepatoma bearing chickens (pI-5.36 and 8.24). A conclusion is drawn that these enzyme forms are apparently present to a greater extent in the hepatoma plasma membrane enriched fractions than in liver membranes and are probably shed into the serum of the tumor bearing animals.     

Serum factors that stimulate fatty acid oxidation: properties of factors

Cultured heart muscle cells, but not HeLa cells, oxidize long-chain fatty acids in medium containing dialyzed serum. Addition of chicken serum dialysate (or non-dialized serum) stimulated palmitic acid oxidation by HeLa cells 10 to 20 fold. This serum activity was not eliminated by lipid extraction, ethanol or acid precipitation, alkaline phosphatase treatment, or autoclaving. About 80% was lost after any one of the following treatments: 6N HCl at 110 degrees C for 16 hr, pepsin, Dowex cation exchange at pH 3, or 1N KOH at 100 degrees C for 1 hr. Serum activity was separated into five or more peaks by gel filtration with Sephadex G-10. Each of these peak fractions was further purified by HPLC using a cyanopropyl-bonded resin. Carnitine, which is important for the transport of long-chain fatty acids into mitochondria for oxidation, also stimulated the oxidation of palmitate. However, these serum factors are not known precursors to carnitine since its immediate precursor 4-n-trimethylaminobutyrate, did not stimulate palmitate oxidation. Total carnitine, including that in acylcarnitine compounds, was approximately 15 microM in the chicken sera to give approximately 0.7 microM in the medium. Based on the fraction of total activity accountable by carnitine and fractional stability to acid, alkali, and pepsin, about 75% of the activity is from non-carnitine compounds. Only one of the factors appears to be carnitine or an acylcarnitine derivative. Several lines of evidence suggest that the other factors are peptide compounds.     

NADPH-cytochrome P450 oxidoreductase from the chicken (Gallus gallus): Sequence characterization,functional expression and kinetic study

Cytochrome P450 monooxygenases have been well known to be responsible for the synthesis of endogenous compounds and the metabolism of exogenous compounds in almost all living organisms, which require NADPH-cytochrome P450 oxidoreductase (POR) as an electron donor to function. In this study, a 2031 bp open reading frame of POR gene was cloned from 35-day-old Roman hen liver, encoding an enzyme of 676 amino acids. Sequence analysis showed that chicken POR shares high homology with other vertebrates PORs and possesses the conserved binding domains of FAD, FMN, and NADPH. The genomic sequences of POR genes from chicken and other four vertebrates have highly conserved exon/intron organization structure. By fusion with bacterial signal peptide, chicken POR gene was functionally expressed in E. coli membrane and showed activities in reduction of cytochrome c and oxidation of NADPH. The Km values for cytochrome c and NADPH were 21.9 ± 2.3 μM and 2.4 ± 0.3 μM respectively. A Ping-Pong mechanism was proposed for chicken POR.     

Activation of phosphodiesterase by chicken iodopsin

Activation of guanosine 3',5'-cyclic monophosphate phosphodiesterase in outer-segment membrane of chicken retina was investigated. Irradiation of dark-adapted chicken outer segment membrane for bleaching of iodopsin increased the enzyme activity twice as much as that in the dark in the presence of GTP. Further irradiation of the sample for bleaching of rhodopsin in the membrane induced some additional activation of the enzyme. However, chicken iodopsin activated the enzyme in frog rod outer segment membrane without irradiation, while chicken rhodopsin did not. Irradiation of chicken iodopsin increased the enzyme activity twice as much as that in the dark.     

Kinetics of the inhibition by naphtholsulfonate compounds of AMP deaminase from chicken erythrocytes

The effect of a variety of naphthalene sulfonate compounds on the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction was analyzed kinetically. Of the naphthalene sulfonate derivatives tested, the compounds with hydroxyl, sulfonate and nitrogen groups such as amino, anilino or azo groups showed an inhibitory effect. The cooperative effect of AMP, analyzed in terms of Hill coefficient, was increased from about 2 to 4 and the maximal velocity was unchanged with the addition of these compounds, suggesting the ligands as an allosteric inhibitor of the enzyme. The inhibition of AMP deaminase by naphtholsulfonate compounds can be qualitatively and quantitatively accounted for by the Monod-Wyman-Changeux model. Theoretical curves yield a satisfactory fit of all experimental saturation and inhibition curves, assuming four binding sites for AMP and the inhibitor, and various KT(I) values. The structure-activity analysis of the interaction of the naphtholsulfonate compounds with AMP deaminase has demonstrated that the affinity of the enzyme for naphtholsulfonates as the inhibitors is correlated with electronic properties of the nitrogen atoms attached to naphthalene moiety: the delocalization of lone electron pair on nitrogen through naphtholsulfonate group makes the compound less basic, resulting in more tight binding of the ligand to the enzyme. Introduction of hydrophobic group to naphtholsulfonate moiety increases the binding affinity for the enzyme, and of the inhibition. These results suggest the location of hydrophobic regions as the allosteric inhibitory sites of the enzyme for the binding of naphtholsulfonate compounds.     

Specific phosphorylation of yeast hexokinase induced by xylose and ATPMg. Properties of the phosphorylated form of the enzyme.

Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit γ globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no cross-reactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 m urea, but was reduced by exposure to 6.0 m urea plus 0.4 m guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.     

Butyrylcholinesterase from Chicken Brain Is Smaller than That from Serum: Its Purification, Glycosylation, and Membrane Association

Applying a new four-step isolation procedure, we have purified butyrylcholinesterase (BChE) from chicken serum to homogeneity with more than 250 U/mg specific activity. The serum enzyme was used for producing monoclonal antibodies. These BChE-specific also recognize BChE from brain, and thus enabled us to isolate the enzymes from embryonic and adult brain that occur only in minute amounts. More than 50% of the brain BChE is membrane-bound. The catalytic and inhibition properties of brain BChE are similar to those of serum BChE. However on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the serum enzyme is represented by a double-band of 79/82 kDa, whereas the brain enzyme has a size of 74 kDa. Limited digestion of the serum and brain preparations by V8-protease leads to similar peptide patterns. Enzymatic deglycosylation shows that their core proteins consist of 59-kDa subunits and that the different molecular weights are due to different glycosylation patterns. The differently sized glycosylation parts of brain and serum BChE may indicate that they subserve different functions. Furthermore, the membrane-bound brain BChE can be solubilized by Pronase or protease K, but not by phosphatidylinositol-specific phospholipase C.     

Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase.

Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Butyrylcholinesterase Is Complexed with Transferrin in Chicken Serum

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.     

The species distribution of xanthine oxidase

1. The distribution of xanthine oxidase in blood and tissues of various animals was studied by means of a radioactive assay capable of detecting 10(-7) unit of enzyme. The method was shown to be applicable to tissues with a high uricase content. 2. Of 16 mammalian species examined, six had low concentrations of xanthine oxidase in the serum. In six non-mammalian species, no activity was detected in the serum. 3. The enzyme was not found in the blood cells of any mammals, but was present in the nucleated red blood corpuscles of chicken, turtle and tortoise. 4. Studies of the tissue distribution in four species demonstrated high activities in the liver and intestinal mucosa and consistently low activities in skeletal muscle, heart and brain. 5. There is a rough correlation between the activity of enzyme in serum and its activity in lung tissue in 12 mammalian species. In the dog, left-atrial blood had higher concentrations of xanthine oxidase than right-atrial blood.     

Thermometric enzyme linked immunosorbent assay: TELISA.

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.     

Distribution in tissues and effects by acute hypoxia on angiotensin I converting enzyme activity in guinea pig and chicken

Angiotensin I converting enzyme activity was measured in homogenates of guinea pig and chicken organs (lung, kidney, heart, ileum, diaphragm and liver), using a spectrophotometric assay for hydrolysis of hippuryl-L-histidyl-L-leucine. High specific activities were found in lung, kidney and diaphragm, but the highest corresponded to guinea pig lung and chicken kidney. Acute hypoxia decreased angiotensin I converting enzyme activity in guinea pig lung and chicken diaphragm, but the changes in kidney were considered non-significant in both the guinea pig and chicken.     

Immunological characterization of bovine lysyl oxidase

Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.     

A preliminary study on viral decontamination of chicken serum using gamma-irradiation

A preliminary experiment was carried out to determine whether a decontamination procedure using gamma irradiation, similar to that adopted in the European guideline for bovine serum contaminated by pestivirus, could be applied to chicken serum. Chicken sera spiked with known amounts of enveloped and non-enveloped chicken viruses were gamma irradiated. The remaining live viruses were then measured by titration and the virus reduction capacity of the irradiation process was established for both enveloped and non-enveloped virus models. In parallel with the irradiation procedure, a classical in vivo extraneous agent test was also evaluated in order to see if it has the capacity to detect low enough levels of live viruses to be used for testing irradiated serum. The results suggest that the principles of the bovine serum decontamination procedure may be applied to chicken serum. Further studies are required to determine if this process would provide an acceptable solution for the viral ‘decontamination’ of chicken serum.