E. G. Buss

The nature of the biochemical lesion in avian renal riboflavinuria III. The isolation and characterization of the riboflavin-binding protein from egg albumen☆

Abstract 1. 1. An autosomal recessive strain of white leghorn hens produces eggs with an insufficient supply of riboflavin because of the absence of the riboflavin-binding protein similar to that reported by Rhodes, Bennett and Feeney . This protein has now been isolated from the whites of eggs of homozygous normal hens by a sequence of adsorptions and elutions from DE-32 cellulose on CM-23 cellulose under controlled pH and ionic strength. 2. 2. The protein has a molecular weight of 32 000 by sedimentation equilibrium measurements. 3. 3. The protein contains 14% carbohydrate with glucosamine, galactose and mannose as the component sugars. 4. 4. The protein is highly cross-linked with eight disulfide bridges. No free sulfhydryl groups are present. 5. 5. The amino acid composition is reported. 6. 6. Tryptophan and an acidic group with a p K a of approx. 3.8 have been implicated in the riboflavin-binding.

The nature of the biochemical lesion in avian renal riboflavinuria—II. The inherited change of a riboflavin-binding protein from blood and eggs☆

Abstract 1. 1. A strain of Single Comb White Leghorn chickens carrying the recessive riboflavinuria alleles (rdrd) was compared with the heterozygote (Rdrd) and the normal genotype (RdRd) in an effort to ascertain the specific effect of the gene. 2. 2. The effect of the mutant gene was observed to cause a deficiency of riboflavin-binding protein in the albumen and yolks and eggs and in the blood serum; this protein has characteristics analogous to the known flavoprotein. 3. 3. The phenotypic expression of this mutant gene is almost exactly 2 (RdRd) : 1 (Rdrd) : 0 (rdrd) in terms of riboflavin-binding proteins in the albumen and yolk.

Triploid-Diploid Mosaic Chicken Embryo

Cytological analysis of an underdeveloped chicken embryo at 6 days of incubation revealed a triploid-diploid mosaic condition. Of the 30 metaphases observed, 19 were triploid and 11 diploid. The triploid cells were 3A-ZZZ and diploid cells 2A-ZZ, as determined for the six largest pairs of chromnosomes.

The role of oligosaccharide in transport of egg yolk riboflavin-binding protein to the egg.

The carbohydrate portion of chicken egg yolk riboflavin-binding protein was examined to determine its role in the biological activity of the protein. Yolk RBP was found to contain 5--6 mannose, five galactose, 12 N-acetylglucosamine and four sialic acid residues. Specific modifications of the oligosaccharide moiety were performed which included removal of sialic acid by mild acid hydrolysis, oxidation of galactose by galactose oxidase, and removal of N-acetylglucosamine and galactose residues by a mixture of glycosidases from Aspergillus niger. All of the modified proteins retained the ability to bind riboflavin although their capacities were lower than that of native yolk RBP. Circular dichroism of the modified yolk RBP samples showed changes in the near ultraviolet, but molar ellipticities in the far ultraviolet displayed only minor variations indicating no gross structural changes. All samples cross-reacted with RBP-specific antiserum. The plasma half-life of 125I-labeled yolk RBP was 62 min. Each of the modified samples was cleared more rapidly from the blood than native yolk RBP. Removal of sialic acid decreased the half-life of yolk RBP by 31%, while the other modifications decreased the half-life by as much as 60%. During a 10-day period following injection of 125I-labeled yolk RBP, 5.9% of the labeled protein was recovered from egg yolk. Relative to native yolk RBP, the transport of asialo-yolk RBP was decreased by 82%. The other modifications resulted in even less transport to the egg, the lowest being glycosidase-treated asialo-yolk RBP which was decreased by over 99%. By comparison of samples with similar clearance times, a positive correlation was made between sialic acid and ovarian transport.

Chemical modifications of riboflavin-binding protein: Effects on function and antigenicity

Abstract Modification of ϵ-amino groups of holo- and apo-flavoproteins was achieved by succinylation, dinitrophenylation, citraconylation and acetamidination. The lysine derivatives with altered charges exhibited significant decreases in native antigenicity and riboflavin binding. Removal of the citraconyl groups resulted in an 80% recovery of antigenicity as well as riboflavin binding. Acetamidination of lysines with the retention of net positive charge indicated that about 4 lysino residues may be involved in native antigenicity but not in riboflavin binding. Apo-flavoprotein treated with 2-nitrophenyl sulfenyl chloride (specific for tryptophan residues) was antigenically analogous to the unmodified protein but completely lost the capacity to bind riboflavin. Additional evidence for the independence of the antigenic site(s) from the riboflavin-binding site came from the fact that the apo-flavoprotein immune complex bound riboflavin and that the holo-protein was antigenic.

Abnormal Water Balance in a Mutant Strain of Chickens

Polydipsia and polyuria are pronounced in chickens of a selected strain and this diabetes insipidus is inherited. The kidneys of such birds are capable of an antidiuretic response when lysine vasopressin or arginine vasotocin is injected. Osmotic pressure and sodium concentration of the plasmas of normal and mutant chickens are identical. Chicks predicted to have diabetes insipidus on the basis of parental pedigree are polydipsic.

The nature of the biochemical lesion in avian renal riboflavinuria—I. Effect of genotype on renal riboflavin metabolism☆

1. 1. A strain of Single Comb White Leghorn chickens bearing the recessive riboflavinuria alleles (rdrd) was compared with the normal genotype (RdRd) in an effort to ascertain the specific effects of the mutant gene. 2. 2. The effect of genotype on the ability of kidney slices to transport riboflavin and p-aminohippuric acid against a concentration gradient was measured. 3. 3. The kidney tissue of both genotypes was assayed for phosphatase, phosphotransferase, flavokinase and FAD pyrophosphorylase. 4. 4. Neither of these lines of investigation revealed any differences between the RdRd and rdrd reproductively active females. 5. 5. On this basis, the reported etiology of riboflavinuria as a renal tubular transport failure is questioned.

Evaluation of commercial mammalian parathyroid hormone (PTH) radioimmunoassay systems for measuring avian PTH

Abstract 1. 1. Five commercial mammalian parathyroid hormone (PTH) radioimmunoassay (RIA) systems were evaluated for their capacity to detect avian PTH. 2. 2. Plasma samples were collected from normal White Leghorn cockerels ( Gallus domesticus ), parathyroidectomized (PTX) cockerels, and cockerels infused intravenously with a calcium chelating agent. 3. 3. None of the commercial PTH-RIA systems revealed significant differences among plasma samples from control, PTX or calcium-chelated cockerels. 4. 4. Some dose-response activity was detected with serial dilutions of chicken and turkey parathyroid gland extracts were tested with the commercial PTH-RIA systems. 5. 5. It is concluded that PTH levels in plasma are too low or avian PTH is too poorly recognized by the RIA antibodies for the commercial mammalian systems to be useful in measuring PTH in avian plasma.

Bone parameters of thick and thin eggshell lines of chickens.

Comparative measurements of bone parameters were made on chickens from thick and thin eggshell lines that were maintained on a normal diet containing 3.5% calcium, injected i.v. with 45CaCl2 and sacrificed 30 min later. There were no significant differences between shell lines for the following measurements on the left femur: specific gravity, ash wt, total calcium, percent calcium and 45Ca content. There was deposition of 45Ca in the femur of all hens--even those in the process of eggshell formation. Skeletal metabolism was not a limiting factor in determining whether a hen produced a thick or thin eggshell.

Effect of carbohydrate modification on transport of chicken egg white riboflavin-binding protein☆

Abstract 1. 1. The role of the oligosaccharide moiety of chicken egg white riboflavin-binding protein (WRBP) was studied by correlating structural modification with physiological effects. 2. 2. Periodate oxidation destroyed much of the carbohydrate moiety but resulted in an inactive protein which could not be used for further physiological studies. 3. 3. Desialylated and galactose oxidase-treated WRBP, were labeled with 125I and used to determine plasma clearance times, uptake by various organs and transport to the egg. 4. 4. The half-life of asialo-WRBP in the plasma was very similar to that of native WRBP although galactose oxidase-treated samples were cleared much more rapidly. 5. 5. Although liver accumulated more WRBP than the other organs examined (ovary and oviduct), only slightly more asialo- or galactose oxidase-treated WRBP accumulated in liver when compared with native WRBP. The amount of galactose oxidase-treated asialo-WRBP in liver was 46% greater. 6. 6. Riboflavin-binding protein isolated from yolks collected over a 10-day period following i injection of 125I-WRBP contained 1.2% of the injected radioactivity. Removal of sialic acid from WRBP decreased transport to the egg to 0.23% and none of the galactose oxidase-treated samples reached the egg within the same time span.