Rhodanese activity in different tissues of the ostrich.

1. The purpose of this investigation was to determine the activity, and compare the pattern of distribution, of rhodanese (thiosulphate: cyanide sulphurtransferase, EC. 2.8.1.1) in different tissues of male and female ostriches. 2. Tissue samples from male and female Blue Neck ostriches were assayed for rhodanese activity by the determination of thiocyanate formed by the action of the enzyme on thiosulphate and KCN. 3. Rhodanese was present in all tissues, and the highest activity was observed in the kidney and liver. Other tissues which contained significant activities of rhodanese were the duodenum, pancreas, heart, caecum and rectum. 4. Unlike other birds, the proventiculus does not appear to have an important role in cyanide detoxification in ostrich and, like mammals, the kidney and liver perform this function. 5. The results suggest that the main organs harbouring high rhodanese activity in the ostrich are associated with sites likely to be required in rhodanese mediated cyanide detoxification.

Differences in the digestive tract characteristics of broiler chickens fed on complete pelleted diet or on whole wheat added to pelleted protein concentrate.

1. The effects of whole grains of wheat on the digestive tract of broiler chickens was studied. A complete pelleted feed was compared with free choice feeding of whole wheat and a pelleted protein concentrate, given from 7 to 29 d of age. 2. Pepsin activity in proventriculus tissue was lower in whole wheat-fed birds than in complete diet-fed birds. The weight (g/kg body weight) of the gizzard was higher in whole wheat-fed birds and its contents had a lower pH. 3. In the intestine, there were no differences in deoxyribonucleic acid (DNA) concentration, protein/DNA, ribonucleic acid (RNA)/DNA, RNA/protein ratios or alkaline phosphatase activity expressed per tissue weight. The weight (g/kg body weight) of the duodenum was lower in whole wheat-fed birds and its contents had a higher pH. Also the activities of alkaline phosphatase and leucine aminopeptidase in the duodenum, and maltase in the ileum, expressed per unit of bird weight, were lower in whole wheat-fed birds. 4. These results suggest that whole grain feeding increases the chemical (pepsin in proventriculus) and physical (gizzard muscle) functionality of the upper part of the digestive tract but decreases the digestive capacity of the intestine. Higher gizzard functionality may play a positive role in the control of bacterial populations. The lower digestive enzyme activities in the intestine may be detrimental in situations of mucosal deterioration caused by intestinal disease.

Stability of porcine and microbial lipases to conditions that approximate the proventriculus of young birds.

In vitro experiments were conducted to characterize the activity and the stability of lipase from animal (crude porcine, CPL; lyophilized porcine, LPL), fungal (Rhizopus arrhizus, RAL; Aspergillus niger, ANL), and bacterial (two Pseudomonas spp., PL1, PL2; and Chromobacterium viscosum, CVL) sources when exposed to conditions associated with the glandular stomach. Activity was measured at pH 3 to 8, 40 C and then monitored in response to temperature (40 C), time of exposure (0 and 30 min), pH (3 and 7), and pepsin level (5, 50, and 500 U/mL). All lipases except ANL and CVL had maximum activity at pH 7 to 8. The optimal pH for ANL and CVL were 5 and 6 to 8, respectively. Exposure of lipases to 40 C and pH 7 for 30 min reduced the activity of all lipases except ANL. In contrast, 40 C increased ANL activity 2.5-fold. Although activity of all lipases was reduced by exposure to pH 3, it was nearly eliminated for CPL and LPL. Pepsin concentration had only minor effects on lipase activity and then only at high concentration. The results demonstrate that bacterial lipases (PL1, PL2, and CVL) and ANL are more stable under conditions that approximate the glandular stomach and may explain why dietary porcine lipase has been ineffective in preventing fat malabsorption in previous in vivo studies.

Stunting syndrome in broilers: effect of age and exogenous amylase and protease on performance, development of the digestive tract, digestive enzyme activity, and apparent digestibility.

Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with stunting syndrome (SS). Naive controls were kept in a parallel room. The chicks were fed a commercial starter diet supplemented with two levels of enzyme preparations to 14 d of age. The experiment was continued to the age of 6 wk in order to estimate compensatory feed intake and growth. In a parallel study, digestibility of the feed was determined from 1 to 3 wk of age with control or inoculated chicks. The enzymes amylase and proteases were produced by Bacillus subtilis and Penicillium emersonii. Enzyme supplementation had no effect on feed intake, growth, or feed utilization, or on digestibility of fat, starch, protein, or energy. Because enzyme supplementation did not consistently affect performance of chicks and no interactions were observed between enzyme supplementation and infection status, data are presented for effects of infection only. Inoculation of SS-infective material reduced performance to 4 wk. Compensatory growth and feed intake were observed from the age of 4 wk onward. At the age of 6 wk the slight retardation of the inoculated chicks was not significant. On Week 1, retention of fat, starch, protein, and energy was significantly depressed in the inoculated chicks. At the age of 2 wk, retention of starch was not depressed, and at the age of 3 wk, the only consistent depression was that observed for fat. The proventriculus weight and content were consistently higher in inoculated chicks, as were the small intestine and intestinal content. The pH of the gizzard content was higher, and that of the small intestine content was lower, in the inoculated birds than in their control counterparts. Stunting syndrome infection was accompanied by a significant depression of trypsin activity in the pancreas at the age of 1 and 2 wk. At these periods, amylase and chymotrypsin were not affected. At 6 wk of age, the activities of amylase, trypsin, and chymotrypsin in the pancreas were higher in the inoculated than in the control birds. In the intestinal chime, amylase, trypsin, and chymotrypsin activities were lower in the inoculated birds on Week 1 and 2 (NS for amylase on Week 1). On Week 6, the activity of all enzymes assayed was higher in the inoculated birds (NS for amylase). It is suggested that the main factors depressing feed intake and growth in SS-infected birds are most probably beyond those of digestion.

Ostrich pepsinogens I and II: purification, activation and chemical and immunochemical characterization of the enzymes from the proventriculus.

Pepsins are a series of gastric proteases secreted as inactive precursors (pepsinogens) which are active at acidic pH. The aim of this study was to purify ostrich pepsin(ogen)s and to compare their biochemical and immunological characteristics with those of pepsin(ogen)s of mammalian and avian origin. Ostrich pepsinogens were purified by ammonium sulphate fractionation, Toyopearl Super Q-650S chromatography and rechromatography, and hydroxylapatite chromatography of a pH 8.0 mucosal extract. Pepsins were obtained through acidification, and purified by chromatography on SP-Sephadex C-50. Amino acid compositions, N-terminal sequences, Ouchterlony double-diffusion as well as Western blot analysis were performed. Two pepsinogens were isolated and purified from the proventriculus of the ostrich, pepsinogens I and II. Both pepsinogens and pepsins were purified to homogeneity as shown by PAGE and SDS-PAGE, with SDS-PAGE revealing M(r) values of 40,400 and 41,900 for pepsinogens I and II, respectively. SDS-PAGE revealed M(r) values of 36,000 and 36,300 for ostrich pepsins I and II, respectively. Ostrich pepsinogens I and II were found to have identical N-terminal sequences, with Asp as N-terminal amino acid. Amino acid compositions were obtained for both pepsinogens, with ostrich pepsinogen I being slightly smaller in size with a total of 356 residues compared to 371 for ostrich pepsinogen II. Pepsinogen II showed a pI of 4.29. Ostrich pepsinogens I and II were found to be immunologically separate entities, and no cross-reactivity was observed between anti-(ostrich pepsinogen I/II) sera and porcine pepsin/pepsinogen. The study indicates that only two pepsinogens are present in the ostrich. They differ in terms of electrophoretic mobility, molecular mass and immunological reactivity, but have been found to have identical N-terminal sequences. It is concluded that both pepsinogens belong to the pepsinogen A class of aspartyl proteases (EC 3.4.23.1).

Molecular adaptation of a leaf-eating bird: stomach lysozyme of the hoatzin.

This report describes a lysozyme expressed at high levels in the stomach of the hoatzin, the only known foregut-fermenting bird. Evolutionary comparison places it among the calcium-binding lysozymes rather than among the conventional types. Conventional lysozymes were recruited as digestive enzymes twice in the evolution of mammalian foregut fermenters, and these independently recruited lysozymes share convergent structural changes attributed to selective pressures in the stomach. Biochemical convergence and parallel amino acid replacements are observed in the hoatzin stomach lysozyme even though it has a different genetic origin from the mammalian examples and has undergone more than 300 million years of independent evolution.

Rhodanese (thiosulfate:cyanide sulfurtransferase) distribution in the digestive tract of chicken.

In this study the level of rhodanese (thiosulfate:cyanide sulfurtransferase) activity in different regions of the digestive tract of chicken was determined and compared with that in the liver, heart, kidney, and lung. All tissues studied contained rhodanese. The highest specific activity of rhodanese was in the submucosal layer of proventriculus, followed by liver, heart, the mucosal layer of cecum, rectum, and kidney. The lowest level was present in lung. These results suggest that in the chicken part of the ingested cyanide is detoxified in the digestive tract, mainly by the proventriculus, and part of the absorbed cyanide is metabolized by hepatic rhodanese.

The isolation and characterization of pepsinogens from the proventriculus of the ostrich Struthio camelus.

Three pepsinogens were isolated and purified from the proventriculus of the ostrich Struthio camelus, by a combination of chromatography steps on DEAE-cellulose, Sephadex G-100 and Hydroxylapatite. The purified pepsinogens manifested peptic activity towards haemoglobin as substrate after activation, but resembled chicken pepsinogens in that they appeared to lose their potential peptic activities during storage. All three pepsinogens contained glycine as N-terminal amino acid, but differed in their overall amino acid compositions. The pH and temperature optima of the activated pepsinogens were determined, as well as their molecular weights.

Carbonic anhydrase localization by light and electron microscopy: a comparison of methods.

The histochemical localization of carbonic anhydrase by Hansson's cobalt-salt method was compared with immunocytochemical localization in the proventriculus (glandular stomach), the chorioallantoic membrane, and in articular and growth-plate cartilages from the domestic hen. Numerous differences were observed. Staining was positive by Hansson's method and negative by immunocytochemistry for the submucosal glands of the proventriculus, articular cartilage cells, resting and proliferating cells of the growth plate, nuclei, and intercellular spaces. Red blood cells stained faintly and inconsistently by Hansson's method. Both methods were in agreement for the cytoplasm of the surface mucosal cells of the proventriculus, the cytoplasm of the villus cavity cells in the chorioallantoic membrane, and in hypertrophic cells of growth-plate cartilage. Acetazolamide usually inhibited the histochemical reaction, even in those sites that, according to other methods, did not contain enzyme. Consequently, acetazolamide inhibition appears to be an unreliable control for the histochemical reaction.

[Immunocytochemical study of the development of pepsinogens in the chick embryo]. / Etude immunocytochimique du développement des pepsinogènes chez l'embryon de Poulet.

The appearance and localization of pepsinogens in the chicken proventriculus during the normal development were studied immunocytochemically with use of the antibodies against an embryo-specific and an adult-specific chicken pepsinogens. The embryonic pepsinogen exists mainly in the apical part of the gland cells and in the secreted substances of 10-20 day embryonic proventriculus. The adult pepsinogen appears during hatching, and all gland cells become positive mainly at the basal portion one day after hatching.

Histochemical localization of carbonic anhydrase in fowl proventriculus.

The carbonic anhydrase activity in fowl proventriculus was studied by the histochemical method of Hanssom. The activity was observed in the mucose membrane cells and in proventricular gland cells. These results, about which there is disagreement in the literature, are discussed in the text.

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail.

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.

Demonstration of acid phosphatase activity in the proventriculus of common weaver bird, Ploceus philippinus and whitebreasted kingfisher, Halcyon smyrnensis.

Acid phosphatase activity was histochemically localized in the proventriculus of two birds namely Ploceus philippinus and Halcyon smyrnensis. It was found that acid phosphatase-rich lysosomal activity appears to be relatively better developed in the proventriculus of piscivorous form, H. smyrnensis than that of granivorous form. P. philippinus. Simultaneously, a possible correlationship between the variable lysosomal activity and specific food diets of the birds has been discussed.

The purification and properties of a single chicken pepsinogen fraction and the pepsin derived from it.

1. Evidence is given for the presence of at least five pepsinogens in a crude extract of mixed chicken stomachs. One of these was purified and could be activated to yield a single pepsin. 2. The molecular weights of the pepsinogen and pepsin were 36000 and 34000 respectively. The pepsin associated at low pH values and low ionic strength. 3. The amino acid analyses of both proteins are given. The pepsin was devoid of phosphate but contained carbohydrate. 4. The N-terminal amino acids of pepsinogen and pepsin were serine and threonine respectively. Five amino acids were released by carboxypeptidase A and it was deduced that serine may be the C-terminal one. 5. Each protein contained one thiol group per molecule as determined by titration with p-chloromercuribenzoate. The rate of the reaction was very rapid with pepsin, but much slower with pepsinogen, although the same group appeared to react in both instances. The enzymic activity of pepsin was unaffected by the modification. 6. The isoionic point of the pepsin was close to pH4.0 and the enzyme was stable for long periods at pH values up to 7.0. 7. The enzyme hydrolysed bisphenyl sulphite almost as rapidly as did pig pepsin A.