Vitamin D in the avian egg. Its molecular identity and mechanism of incorporation into yolk.

The chemical identity of vitamin D in the egg of the domestic fowl was studied by analysing radioactivity in eggs from hens injected with [3H]cholecalciferol. Labelled molecules were found throughout the egg, but the concentration of total radioactivity in albumin was only 5-7% of that in yolk. In lipid extracts of yolk, more than 90% of the radioactivity was as unchanged cholecalciferol and 5% as 25-hydroxycholecalciferol. Only about 3% of the radioactivity in albumin was chloroform-soluble, and of this 40% was 25-hydroxycholecalciferol and 15% was cholecalciferol. Evidence is presented to support the idea that the specific transport of cholecalciferol into yolk is mediated by a cholecalciferol-binding protein in blood. This protein forms a complex with yolk proteins in transit from liver to ovary via the blood. A cholecalciferol-binding protein, chromatographically similar to that from blood, was found in egg yolk. It is postulated that cholecalciferol forms part of a complex with its specific binding protein, Ca2+ and the yolk phosphoprotein, phosvitin. This complex is then incorporated into yolk by the thecal cells of the ovarian follicle.     

25-Hydroxycholecalciferol-binding protein: Partial purification from rat duodenal mucosa cells

The 25-hydroxycholecalciferol-binding protein has been partially purified (purification factor: 37) from rat duodenal cytosol, using chromatographic procedures on gel and ionic exchange resin. This partially purified protein bound 25-hydroxycholecalciferol with high affinity (K_D = 5.7 × 10^?9M) and low binding capacity (23 × 10^?12 mole/mg of protein. Using a rabbit antiserum obtained against such partially purified protein, we demonstrated that 25-hydroxycholecalciferol cytosolic binder and 25-hydroxycholecalciferol plasmatic binding share common antigenic sites.     

Binding of aminoacyl-tRNA to rat liver ribosomal proteins

Rat liver ribosome treatment with ethanol and 1 M NH₄Cl releases some 31–33 ribosomal proteins. This split protein fraction binds Phe-tRNA, Ac-Phe-tRNA, Met-tRNA_M and f-Met-tRNA_F in the absence of K⁺ and Mg⁺⁺ ions. When the split protein fraction is passed through Sephadex G-100 only six proteins are retained in the column: S10, S14, S15, S19, L35, and L36. The aminoacyl-tRNA binding activity of this protein fraction retained in the Sephadex G-100 column is similar to that of the total split protein fraction, suggesting that the above six proteins, or only some of them, are involved in the binding reaction.     

Some characteristics of new tissue-binding proteins for metabolites of vitamin D other than 1,25-dihydroxyvitamin D

Protein(s) have been found in a wide range of tissues which have a high affinity for 25-hydroxycholecalciferol. Of the tissues examined only erythrocytes do not have this protein. The properties of the protein have been examined and it has been found that the association constants range from 2 · 10⁹ to 5 · 10⁹ M⁻¹ and the sedimentation constants between 5.0 and 6.0 S. It was not possible to distinguish the proteins from the different tissues by their S values, mobility on gel electrophoresis or behaviour on ion-exchange chromatography. These techniques were all used, however, to show that the tissue 25-hydroxycholecalciferol binding protein is distinct from the main plasma binding protein for this steroid and from the intestinal 1,25-dihydroxycholecalciferol-binding protein. A protein has been found in the plasma of rachitic animals but not of normals, which is apparently indistinguishable from this new tissue 25-hydroxycholecalciferol-binding protein. The steroid specificity of this new binding protein has been shown to be dependent upon a C-25 hydroxyl group, and an intact conjugated double bond system. Possible functions for this protein have been briefly discussed.     

Comparative survey of blood thyroxine binding proteins in turtles

The nature of plasma thyroxine (T4) binding activity was surveyed in turtles; binding to [125I]T4 was measured on polyacrylamide gel electrophoresis--PAGE--and on minicolumns of Sephadex G-25. An electrophoretically distinct T4 binding protein was identified in all 8 species of Pseudemys studied and in 3 other genera (Chrysemys, Deirochelys, and Emyoidea) of the same family, Emydidae. Levels of this binding activity were highly variable among individuals, but they consistently showed a similar low relative mobility (Rf) compared to albumin, and a relatively low capacity was indicated by displacement with unlabeled T4. Two emydids (Terrapene, Clemmys) showed a similar slow migrating binding peak, but binding activity was low and not as easily displaced by unlabeled T4. T4 binding to albumins was minimal in most of these emydid species, even when binding to the higher affinity, low capacity component was low or displaced by unlabeled T4 (2.5 micrograms/ml). In contrast, there was no clear evidence for a similar high affinity, low capacity binding protein in any of the other 19 species representing 13 genera of 8 families from two suborders. In these species, binding activity on Sephadex G-25 was typically low and binding on PAGE was associated largely with albumin; binding levels for albumins were highly variable. In several nonemydids (from distant lineages), binding activity on Sephadex was elevated and PAGE showed a second binding protein distinct from albumin, but it had high capacity (not readily saturable). Thus, an evolutionary divergence in T4 transport proteins is suggested within Chelonia.     

Vitamin D metabolite-binding proteins in human tissue

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD₃) and 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD₃-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport α-globulin. A cathodal, 1,25-(OH)₂D₃-binding protein, which sedimented at 3–4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.     

Comparative role of proteins in transport of HCH-isomers in desert locust, Schistocerca gregaria Forskal and bovines

The studies on binding of hexachlorocyclohexane (HCH) with carrier proteins were carried out to establish the role of proteins in the transport of insecticides in insects. Sephadex G-200 column chromatography resolved haemolymph of adult male desert locust, Schistocerca gregaria into three major protein peaks. There was significant binding of gamma-HCH with first protein peak (F1). Two classes of binding sites were observed on first protein peak for gamma-HCH. However low level of binding was observed with the third protein peak (F3) of the haemolymph. Bindings of HCH-isomers (alpha, beta and gamma) with bovine serum albumin (BSA) were not related to their water solubilities. Moderate to low affinities (1.4 -1.84 x 10(6) M(-1)) of HCH-isomers for BSA were observed. The present studies showed that more HCH binds to haemolymph lipoprotein of locust as compared to BSA. This indicates a significant role of haemolymph proteins in the transport of insecticides in insects.     

Gibberellic acid—Binding proteins from pea stems

Summary The formation of complexes of gibberellic acid (GA₃) and proteins under in vitro conditions was studied. It was shown that labelled GA₃ binds to soluble cytoplasmic proteins, although a considerable amount of radioactivity remains in the pellet containing nuclei and cell debris. GA₃-protein complexes are excluded from Sephadex G-10 column with the void volume. They sediment in linear sucrose density gradients as three distinct peaks, having higher S values than bovine serum albumin, used as a marker. Soluble GA₃-protein complexes can be separated into four zones of radioactivity upon ion exchange chromatography on DEAE-Sephadex A-50 column, each of them eluting with a different KCl concentration. Agarose gel electrophoresis of GA₃-protein complexes reveals two zones of radioactivity at the anodic part of the electrophoretogram. After extraction of the complex with ethanol, more than 90% of radioactivity is found in the ethanolic phase, which indicates that the binding is not covalent. GA₄₊₇ and GA₁₃ decrease the binding of GA₃ to cytoplasmic proteins for 30%, suggesting that some common binding sites exist at the same binding proteins.Abbreviations BAP benzylaldenine - GA gibberellic acid - IAA indolyl-3-acetic acid - TSS tris-sucrose-salts     

Human serum binding protein for vitamin D and its metabolites (DBP): evidence that actin is the DBP binding component in human skeletal muscle

The tissue protein which tightly binds the human serum binding protein for vitamin D and its metabolites (HDBP) was studied in soluble extracts of human skeletal muscle. A tissue protein-HDBP complex was effected in vitro by the addition of human serum Cohn IV to high-speed supernatant from muscle, and the complex was partially purified by ion-exchange chromatography, gel filtration, and affinity chromatography. The faster-sedimenting complex was retained longer than HDBP on DEAE-Sephacel columns, and was estimated to have a size of 100,000 daltons by gel filtration. The complex displayed inhibitory activity to deoxyribonuclease I (DNase I), whereas HDBP alone did not. When the complex was applied to affinity chromatography columns, immunoassayable HDBP was retained by DNase I-agarose and two dominant proteins of ~58,000 and 45,000 M_r were retained by the IgG fraction of anti-HDBP serum covalently bonded to amino-agarose, as revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis. Pure HDBP does not bind to nor inhibit DNase I, but an actin-HDBP complex does. These data suggested that the tissue component with high affinity for HDBP was actin. Incubation of equimolar amounts of polymerized actin and pure HDBP in its apo form resulted in the depolymerization of the actin. This depolymerizing activity was also observed with HDBP saturated with cholecalciferol, 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol, or 1,25-dihydroxycholecalciferol.     

Binding properties of serum vitamin D transport proteins in vertebrates for 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol in vitro.

1. The affinities of the specific vitamin D plasma transport proteins for 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol were studied in 34 vertebrate species. 2. Fish plasma proteins bound 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol with equal efficiency. 3. Vitamin D transport proteins in birds and a monotreme bound 25-hydroxycholecalciferol more efficiently than 24R, 25-dihydroxycholecalciferol; in one bird the two seco-steroids were bound with equal efficiency. 4. Transport proteins from marsupial and placental mammals bound 24R, 25-dihydroxycholecalciferol more efficiently than 24S, 25-dihydroxycholecalciferol. 5. Twelve mammal transport proteins bound 25-hydroxycholecalciferol and 24R, 25-dihydroxycholecalciferol with equal efficiency, however, in six mammals 25-hydroxycholecalciferol was more efficiently bound.     

The folate and thiamine transport proteins of lactobacillus casei

Two separate binding proteins, one specific for folate and the other for thiamine, have been isolated from membrane fragments of Lactobacillus casei. Purification to homogeneity was achieved by fractionation of the Triton-solubilized proteins with microgranular silica (Quso G-32) and Sephadex G-150. Amino acid analyses revealed that the folate (M_r = 25,000) and thiamine (M_r = 29,000) binders have unusually low polarity constants, 0.32 and 0.26, respectively. Evidence obtained with intact cells has established a direct role for these binding proteins in transport of the corresponding vitamins: (A) In each case, the processes of binding and transport showed similarities in substrate affinities and repression by excess vitamin in the growth medium. (B) Competition studies employing amethopterin, 5-formyl tetrahydrofolate, and 5-methyl tetrahydrofolate (for folate) and thiamine monophosphate and thiamine pyrophosphate (for thiamine) have shown that the ability of these compounds to inhibit the transport of the corresponding vitamins is paralleled by their ability to inhibit binding. (C) Amethopterin-resistant mutants which are defective in folate transport have a comparable defect in ability to bind folate. (D) Amethopterin-resistant cells which (compared with the parent cell line) contain folate transport systems with altered affinities for amethopterin also contain binding proteins whose affinities for amethopterin have changed by equivalent amounts. (E) Both the transport and binding of folate by one of the mutants were stimulated (approximately 3-fold) in parallel by the addition of mercaptoethanol.     

Selenium binding proteins in rat tissues

The 100,000 × g extracts of rat intestine and colon were incubated $\text{in}\text{vitro}$ with Na₂[⁷⁵Se]O₃. Chromatography of this material on a Sephadex G-100 column produced three radioactive peaks corresponding to molecular weights of 17,000, 68,000 and > 90,000. The 17,000 peak corresponded to a protein which sedimented in the 2S region of a 5–20% ($\text{w}\text{v}$) linear sucrose density gradient. Selenium binding to this protein was specific, stable and sensitive to thiol inhibitors such as p-chloromercuriphenylsulfonic acid (1 mM) and iodoacetamide (2 mM). Chromatography of rat serum - [⁷⁵Se] complex on Sephadex G-100 yielded only two radioactive peaks that corresponded to molecular weights of 68,000 and > 90,000. The 2S selenium binding protein of intestine and colon may mediate the biological functions of selenium in those tissues.     

Reference concentrations of cholecalciferol in animals: a basis for establishing non-target exposure

Abstract

Cholecalciferol (vitamin D₃) is widely used as a vertebrate pesticide in New Zealand. However, cholecalciferol also occurs naturally in animals. Therefore, when trying to determine whether a non-target animal has been exposed to cholecalciferol baits, knowledge of the baseline cholecalciferol concentrations in the animal's plasma and tissue is required. We analysed cattle, sheep, pig, deer, dog and cat plasma and liver samples for the vitamin D₃ metabolite 25-hydroxycholecalciferol (25-OHD), a sensitive biomarker for cholecalciferol. Based on these data and a literature search we present 25-OHD reference ranges. We also examined the literature for 25-OHD concentrations in poisoned animals and compared these to the reference ranges. Where plasma and liver samples have 25-OHD concentrations at least four times higher than our reference ranges it is likely that the animal has been exposed to cholecalciferol baits. 25-OHD concentrations 10 times higher than the reference range indicate ingestion of abnormally high amounts of cholecalciferol.     

Comparison of media proteins from ovariectomized rat uteri following estrogen treatment

Ovariectomized rats were treated with estradiol for 3 days after which their uteri were incubated in vitro and radioactive media proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Media were also chromatographed on G-25 Sephadex Blue Sepharose columns to isolate subsets of proteins. The results demonstrate that two proteins are consistently increased following estrogen treatment. These proteins have molecular weights of 104,000 and 65,000. Neither protein binds to Blue Sepharose to a great extent. The use of the protein synthesis inhibitors, emetine and actinomycin D, demonstrates that the proteins are synthesized de novo. These two proteins may serve as markers for genomic response to estradiol in the rat uterus.     

Comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites

The comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites were investigated to evaluate: (1) optimization of sensitivity (2) crossreactivity of these compounds in their respective assays and (3) side chain steric requirements of the vitamin D molecule for optimum binding to the calciferol binding protein or bovine thymus receptor. Disequilibrium assay conditions improved assay sensitivity 30-fold for the calciferol assay and approx 3-fold for metabolites in the 25-hydroxycalciferol and 1,25-dihydroxycalciferol assays. Ergocalciferol compounds were uniformly less efficient in their association with the proteins tested than were their cholecalciferol counterparts, with one exception. In the calciferol assay, cholecalciferol had greater affinity for the the calciferol binding protein than did ergocalciferol. In the 25-hydroxycalciferol assay affinity for the calciferol binding protein was 25-hydroxycholecalciferol = 24,25-dihydroxycholecalciferol greater than 25-hydroxyergocalciferol greater than 25S,26-dihydroxycholecalciferol greater than 24,25-dihydroxyergocalciferol greater than 25,26-dihydroxyergocalciferol. In the assay for 1,25-dihydroxycalciferol, bovine thymus receptor recognized 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol equally. From the forthcoming data it appears that hydroxyl and/or methyl groups on the calciferol side chain alter the ability of these physiological compounds to associate with the calciferol binding protein.     

The gel-filtration behaviour of proteins related to their molecular weights over a wide range

1. Correlation between elution volume, V_e, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (α-crystallin) on Sephadex G-200 columns at pH7·5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V_e–log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V_e–log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of `typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including γ-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10⁶. 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of γ-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

The synthesis and turnover of rat liver peroxisomes. I. Fractionation of peroxisome proteins

Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D-amino acid oxidase, L-α-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D-amino acid oxidase, and L-α-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.     

Incorportion of oxygen-18 into the 25-position of cholecalciferol by hepatic cholecalciferol 25-hydroxylase.

The oxygen enzymically inserted as a hydroxy function by rat liver post-mitochondrial fraction into the 25-position of cholecalciferol to giver 25-hydroxycholecaliferol is derived exclusively from molecular O2. Therefore like the other two cholecalciferol hydroxylases, i.e. 25-hydroxycholecalciferol 1alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, the cholecalciferol 25-hydroxylase is also a mono-oxygenase ('mixed-function oxidase').     

Purification and Some Properties of Amylase Inhibitor (S-AI)

Amylase inhibitor (S-AI) was purified by about 25 times from culture filtrate of Streptomyces diastaticus subsp. amylostaticus No. 2476 through the methods of adsorption on active carbon, column chromatographies on Dowex 50 W × 2 (H-form) and Dowex 50 W × 2 (NH₄-form), gel filtration on Sephadex G-25, El-complex formation with BLA, isolation of complex by gel filtration on Sephadex G-75, dissociation from complex by the method of acid denaturation, rechromatographies on Dowex 50 W × 2 (NH₄-form) and Sephadex G-25. Homogeneity of this S-AI was examined by means of TLC, where S-AI gave a single spot in various solvent systems. S-AI specially inhibited α-amylases and glucoamylase, but not β-amylases and other glucoside hydrolases.

S-AI was a very stable substance, as it retained 100% of its original activity after being kept for 30 min at 100°C in a pH range between 3.0 and 10.0. The molecular weight of S-AI was estimated to be about 1500 by gel filtration on Sephadex G-15.

S-AI was regarded to be an oligosaccharide which was mainly composed of glucose in an amount of about 85 %. S-AI was hydrolyzed by β-amylase from non-reducing terminal and released two moles of maltose succesively.