Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum

Cattle and water buffalo sera labelled with vitamin D₃[¹⁴C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin.
Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.     

Effect of pH on vitamin B12 binding by transcobalamins

An investigation has been made of the effect of varying pH at constant ionic strength on vitamin B₁₂ binding by human serum and by two transcbalamin fractions separated from serum by gel filtration. It was found that the methodology used had a considerable influence on the results obtained. Genuine effects of pH were largely confined to reduced vitamin B₁₂ binding at very acid and very alkaline pH. However, due to an adsorption artefact involving transcobalamin II, certain methods appeared to demonstrate a marked decrease in vitamin B₁₂ binding between pH 4.5 and pH 10.3, especially in the range of pH 5.3–7.5.     

Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum.

Cattle and water buffalo sera labelled with vitamin D3[14C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin. Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.     

Vitamin B12 enhances the stimulation of DNA synthesis by serum in resting cultures of 3T6 cells

When low levels of serum are used to stimulate resting cultures of 3T6 cells the limiting factor in increasing DNA synthesis is a low molecular weight component, rather than a polypeptide growth factor. The former component is absent from Dulbecco's modified Eagle's medium (DEM) but present in Waymouth medium. We identify the essential component as vitamin B₁₂. Addition of vitamin B₁₂ to DEM increases the effectiveness of low levels of serum, as assessed both by [³H]TdR incorporation and autoradiography. Thus cultures of 3T6 cells in vitamin B₁₂-supplemented DEM offer a useful in vitro system for testing the mitogenic potency of polypeptides and serum fractions.     

Evidence for 15 genetically determined electrophoretic variants of transcobalamin II in rabbit serum

By starch gel electrophoresis and autoradiography two classes of vitamin B₁₂ binding proteins were detected in rabbit serum. By analogy to the nomenclature used in man, the two classes of proteins were named transcobalamin I (TCI) and transcobalamin II (TCII). Fifteen TCII phenotypes were observed, and family data indicated that they are controlled by five allelic codominant genes. The possibility that the five genes arise from the action of at least two polymorphic and closely linked structural loci is discussed.This work was supported by a grant from the Consiglio Nazionale delle Ricerche (Contract No. 78.00223.80).     

Genetic polymorphism of human plasma α1-B-glycoprotein: phenotyping by immunoblotting or by a simple method of 2-D electrophoresis

Summary Genetic polymorphism of human plasma (serum) ₁B-glycoprotein (₁B) was observed using one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 of plasma samples followed by Western blotting with specific antiserum to ₁B. A simple method of two-dimensional agarose gel electrophoresis (pH 5.4) — horizontal PAGE (pH 9.0) of plasma samples, followed by general protein staining, was reported as an alternative method for ₁B typing. The there different phenotypes of ₁B observed (designated 1-1, 1-2, and 2-2) were apparently identical to those reported by Altland et al. (1983), who used double one-dimensional electrophoresis. Family data supported the hypothesis that the three ₁B phenotypes are determined by two codominant alleles at an autosomal locus, designated A1B. Allele frequencies in a Swedish population were: A1B ^*1, 0.937; A1B ^*2, 0.063; PIC, 0.111. For clues on linkage relationships of human A/B, the previously known linkages of A1B in pigs and horses, including the one between A1B and the gene that determines susceptibility to malignant hyperthermia in pigs were discussed.     

Serum albumin and erythrocyte adenosine deaminase polymorphisms in Asian macaques with special reference to taxonomic relationships amongMacaca assamensis,M. radiata,andM. mulatta

Serum albumin (Alb) and erythrocyte adenosine deaminase (ADA) polymorphisms in Asian macaques were investigated by means of starch gel electrophoresis. The materials comprise a total of 2,323 blood samples from eight species, namely,Macaca fuscata,M. mulatta,M. cyclopis,M. fascicularis,M. nemestrina,M. speciosa,M. radiata, andM. assamensis. It was observed that three Alb phenotypes were controlled by two codominant alleles, Alb _mac ^A and Alb _mac ^B and six ADA phenotypes by four codominant alleles, ADA _mac ¹ , ADA _mac ² , ADA _mac ³ , and ADA _mac ⁴ . The taxonomic relationships amongM. assamensis,M. radiata, andM. mulatta were analyzed by measuring theNei's (1975) genetic distance. The result supportedHill andBernstein's (1969) postulation thatM. assamensis was more closely related phylogenetically toM. radiata than toM. mulatta.     

Effects of Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes

Abstract: We have suggested recently the existence of three subtypes of B₂ bradykinin receptors in tissues of guinea pigs. We have classified these B₂ bradykinin receptors into B_2a, B_2b, and B_2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [³H]bradykinin to the B₂ subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B_2a and B_2b subtypes, specific [³H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl-5′-yl-imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp³]-bradykinin, which shows approximately 20-fold greater selectivity for the B_2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B_2a subtype, which has higher affinity for [Hyp³]-bradykinin and bradykinin than the B_2b subtype, was lost in the presence of GppNHp, whereas binding to the B_2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [³H]bradykinin binding to B_2b and B_2c subtypes in lung. [³H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B_2a bradykinin subtype is coupled to G proteins. The B_2b and B_2c subtypes are either not coupled to G proteins, or may be coupled to the G^o-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B₂-type bradykinin receptors in guinea pig.     

Marine Brown Algae Requiring Vitamin B12

The brown algae Lithosiphon pusillus, Ectocarpus fasciculatus and Pylaiella litoralis were cultivated in bacteria-free cultures in artificial sea water, ASP 6F. The growth was tested with different additions of vitamins and other metabolites. Lithosiphon pusillus and Ectocarpus fasciculatus were found to require vitamin B₁₂ for optimal growth. The zoospores of Pylaiella litoralis, when cultured in medium Asp 6F with kinetin added, had an absolute requirement for vitamin B₁₂.     

Polymorphism in a casein fraction of sows' milk

Polyacrylamide vertical gel electrophoresis was used to separate the caseins of sows' milk. Polymorphism was found in a region of the gels designated Cn₃. The Cn₃ polymorphism consists of two bands which appear to be controlled by two codominant alleles designated Cn ₃ ^A and Cn ₃ ^B . Homozygotes possess one band and heterozygotes posses both bands of equal intensity giving the following phenotypes: Cn₃A, Cn₃AB, and Cn₃B. A chi-square test for goodness of fit of the observed phenotypes to that expected by the Hardy-Weinberg equilibrium formula and a segregation analysis of eight matings involving 19 female progeny conform to the hypothesis that the Cn₃ polymorphism is controlled by two codominant autosomal alleles. Further family studies will be necessary to confirm the genetic control of the Cn₃ polymorphism.Deceased.     

Enhancement of chemiluminescence for vitamin B12 analysis

In the current article, chemiluminescence (CL) from the vitamin B₁₂ and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B₁₂ using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B₁₂ in vitamin B₁₂ tablets, multivitamin capsules, and vitamin B₁₂ injections. Experimental parameters were optimized, including luminol concentration, urea-hydrogen peroxide (urea-H₂O₂) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5 pg/ml, and the linear range was 10 pg/ml to 1 μg/ml with a regression coefficient of R² = 0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea-H₂O₂ in the presence of vitamin B₁₂. Extraction of vitamin B₁₂ was carried out, and the observed recovery was 97-99.2% with a relative standard deviation in the range of 0.30-1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.     

Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

The C-terminus type I collagen is a major binding site for heparin

The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15–1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TC^B was shown to have greater binding affinity for heparin than the N-terminal TC^A fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind.     

Bile and detergent interaction with the radioassay for vitamin B12 binders using protein and dextran-covered charcoal

Bile acids and detergents such as Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate interfered with the radioassay for vitamin B₁₂ binders. The effect was dependent on the concentration of the detergent. The detergents and bile acids were found, by use of [¹⁴C]glycocholic acid, to bind firmly to the covered charcoal in the assay, thereby blocking the binding of free vitamin B₁₂ to the charcoal. An increased ⁵⁷Co-labeled vitamin B₁₂ concentration was therefore observed in the final supernatant in the assay, giving rise to an apparent but false vitamin B₁₂-binding capacity.     

The kinetics of intrinsic factor-vitamin B12 binding

The kinetic behaviour of intrinsic factor-vitamin B₁₂ binding has been examined under varying conditions using an albuminised charcoal separation technique. The overall reaction obeys second order rate laws. The intrinsic factor considered alone obeys first order laws; the velocity of reaction of vitamin B₁₂ is too fast for measurement by the technique described but by deduction obeys first order laws. Rate constants as three temperatures, (k₂ at 25°C=1.56·10⁸·mole^?1·s^?1) the activation energy (E=12.7 kJ·mole^?1) and Arrhenius constant (A=2.7·10¹⁰ 1·mole^?1·s^?1 have been calculated. There is the possibility of diffusion control of the reaction in which case the E and A values are invalid. The effect of pH on the reaction has been studied and the results discussed in relation to the pH studies of other workers whose results show disagreement. Albumin coated charcoal was shown to discriminate between intrinsic factor-vitamin B₁₂ and free vitamn B₁₂ over a wide pH range. The apparent under-estimation of intrinsic factor in dilute solution was shown to be due to adsorption of the intrinsic factor to plastic tubes.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

Holotranscobalamins in B12 and non B12 requiring prokaryotes and eukaryotes

Transcobalamins, vitamin B₁₂ binding proteins, deliver B₁₂ to cell surface receptors which then permit B₁₂ to cross cell membranes for metabolic use. There is little documentation concerning B₁₂ binding proteins in bacteria and protists. We found that prokaryotes and eukaryotes requiring B₁₂, as well as those protists synthesizing B₁₂, also produce several transcobalamins for functionally transporting B₁₂ similar to humans.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.