An antiovulatory decapeptide of higher potency which has an L-amino acid (Ac-Pro) in position 1.

Ac-[Pro¹, $\text{D}\text{-}\text{Phe}{}^2$, $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH completely inhibited ovulation in cycling rats at 200μg/rat and is comparable in activity to the corresponding D-1-analogue. This Ac-Pro¹-analogue is the most potent antiovulatory peptide yet known having an $\text{L}$-amino acid residue in position 1. This result shows that for the design of potent inhibitors of ovulation, a $\text{D}$-amino acid residue is not essential in position 1. The corresponding Ac-$\text{D}$-Pro¹- and Kic¹-analogues completely inhibited ovulation at 750μg/rat, but not at 200μg/rat, and the Cpc¹-analogue was inactive at these dosages.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

A fluorimetric study of Mg2]nduced structural changes in thylakoid membrane protein.

Low concentrations of Mg²⁺ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant K_D = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency T_max = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation $\text{R = 17.3}\text{A}\text{?}$ is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg²⁺ binds to the delipidated membranes with a dissociation constant K_D = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg²⁺ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.     

Probing of the porcine serum lipoprotein surfaces by Mn(II) binding: an e.s.r. study

Mn(II) ions were used for probing the surfaces of porcine LDL₁, LDL₂ and HDL. From the intensity of the e.p.r. lines corresponding to the unbound Mn(II) the percentage of the ions bound to the lipoprotein surface is determined. From the titration curves the binding parameters, dissociation constant. K_d, and the number of binding sites, n, in all the three lipoproteins studied have been derived. There are at least two types of binding sites in each lipoprotein class. The ”weak’ binding sites are charaterized by approximately the same value of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(≈ 6.2 × 10}{}^{\mathrm{?3}}\text{mol l}{}^{\mathrm{?1}}$ and different values for n (n = 114 for LDL₁, n = 135 for LDL₂ and n = 28 for HDL). Similarly, for the ”strong’ binding sites K_d ≈ 1.6 × 10^?4 mol l^?1 and the number of binding sites is 15, 20 and 5 for LDL₁, LDL₂ and HDL respectively. It is concluded that the binding sites are probably located in the protein part of the lipoproteins and that they are mainly associated with the negatively charged amino acids.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

Determination of the rates of synthesis and degradation of vitamin D-dependent chick intestinal and renal calcium-binding proteins

Vitamin D₃ and its biologically active metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D₃-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D₃ or 1,25(OH)₂D₃ to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 29}\text{h}$) and that for renal CaBP was 0.019 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 36}\text{h}$) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH)₂D₃ to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH)₂D₃. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D₃-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH)₂D₃ to each target organ.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

An hydroxylapatite batch assay for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor complexes.

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D₃-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, $\text{v}\text{v}$) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D₃ receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D₃ and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.     

Electron paramagnetic resonance study on calmodulin: conformational change and interaction with divalent cations

Biologically active spin labelled derivatives of calmodulin were prepared and used to study CA²⁺- and Mg²⁺-induced conformational changes of the protein. The rotational correlation time of the spin labelled residues increased upon addition of divalent cations. Two calcium ions per spin labelled calmodulin were found to induce a 75% conformational change, whereas four calcium ions were necessary for a maximum conformational change. The increase in rotational correlation time induced by Mg²⁺ is less pronounced. Two different covalently attached spin labels (iodoacetamide and maleimide) were compared and marked differences were found in their chemical stability. The binding of manganese ions to calmodulin could be observed directly from the electron paramagnetic resonance spectra of these paramagnetic ions. Two specific classes of binding sites, each binding two manganese ions with $\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.6 × 10}{}^{\mathrm{?6}}\text{m}\text{and}\text{k}{}_{\mathrm{D}}\text{= 3 × 10}{}^{\mathrm{?5}}\text{m}$, respectively, were determined. Further ion binding occurs at non-specific sites.     

D2O-alanine exchange reactions catalyzed by alanine racemase and glutamic pyruvic transminase

NMR studies in D₂O (>90%) reveal that Alanine Racemase (5.1.1.1.) from $\text{B. subtilis}$ catalyzes the exchange of the α hydrogen of $\text{D}$- and $\text{L}$-alanine with D₂O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of $\text{L}$-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of $\text{L}$-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.     

The effects of some cations and anions on spin labeled cytoplasmic membranes of bacillus subtilis

The effects of CaCl₂, MgCl₂, LaCl₃ and some alkali halides on $\text{Bacillus subtilis}$ cytoplasmic membranes were studied using stearic acid spin labels. The results indicate that two mechanisms are operating when these ions interact with $\text{B. subtilis}$ membranes. At low ionic concentrations (0 to 0.1 M) there is direct cation binding to the anionic membranes which in the case of Ca²⁺, Mg²⁺ and La³⁺, confers rigidity on the membranes and reaches saturation when the number of cations present equals the number of anionic sites on the lipids. At high concentrations there is a further effect on the membranes that parallels the known organizing/disorganizing effects that the ions studied have on water structure.     

A soybean lectin having 4-O-Methyl-D-Glucuronic acid specificity

Evidence is presented for the presence of a new lectin activity in soybean seeds [$\text{Glycine}\text{max}$ (L.) Merrill] that has specificity towards the 4-O-methyl-$\text{D}$-glucurono-$\text{L}$-rhamnan exopolysaccharide produced by certain strains of $\text{Rhizobium}\text{japonicum}$. Bacterial agglutination and precipitin reactions revealed the lectin activity in phosphate-buffered saline extracts of seeds of all cultivars tested, including the “lectinless” varieties. Reaction of such extracts with carbohydrate haptens demonstrated that the specificity of the binding was towards 4-$\text{O}$-methyl-$\text{D}$-glucuronic acid, $\text{D}$-glucuronic acid and their methyl glycosides.     

Identification of a structural hairpin in the filamentous chimeric phage M13Gori1

Crystals of alkaline phosphatase (EC 3.1.3.1; M_r 94,000) grown at pH 9.5 from 2.25 m-(NH₄)₂SO₄ with 5 × 10^?5 m-Zn and 10^?2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH₄)₂SO₄ with 10^?2 m-Zn and 10^?2 m-Mg present. The crystals are orthorhombic with $\text{a = 195.5}\text{A}\text{?}\text{, b = 168.3}\text{A}\text{?}\text{and}\text{c = 76.33}\text{A}\text{?}$, and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K₂PtCl₄, KAu(CN)₂ and K₂OsO₄ derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major $\text{α}\text{β}$ domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the $\text{α}\text{β}$ domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the $\text{α}\text{β}$ domain. In both there is extensive monomer-monomer contact.Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10^?2 m or cadmium at 10^?2 or 5 × 10^?2 m reveals seven metal binding sites per dimer. The active centers are 32 Å apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 Å. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 Å away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 Å of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10^?2 m-Cd two metal sites (D and D′) are found 25.5 Å from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 Å. The seventh Cd site, E, is 20 Å from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate.The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10^?3 m, 1.5 × 10^?1 m and 1.3 × 10^?2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.     

Structure and properties of hemocyanins. XIII. Dissociation of Helix pomatia alpha-hemocyanin at alkaline pH

Helix pomatia α-hemocyanin can dissociate stepwise into $\text{1}\text{2}$-size, $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules. Both $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules can occur in two isomeric forms, differing considerably in sedimentation behaviour.The effects of pH, ionic strength, temperature, Ca²⁺ concentration and oxygen pressure on these dissociation and isomerization steps were investigated systematically by sedimentation analysis.Dissociation and isomerization are favoured by increasing pH or temperature. Changes in ionic strength affect each step differently. Calcium ions are extremely effective in preventing dissociation and isomerization at low ionic strength, but this stabilizing ability diminishes at higher ionic strengths. Oxygen binding shifts the pH-dependent dissociation of whole into $\text{1}\text{2}$-size molecules to higher pH. Oxygen has no effect on the other dissociation steps. Intermolecular interactions appear to be predominantly electrostatic.     

The site of 1α,25-dihydroxyvitamin D3 production in pregnancy

Metabolism of 25-hydroxyvitamin D₃ (25-OH-D₃) in pregnancy was investigated $\text{in}\text{vitro}$ in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [³H]-25-OH-D₃. The homogenates from fetuses produced significant amounts of $\text{[}{}^3\text{H]-1α,25-dihydroxyvitamin}$ D₃ [1α,25-(OH)₂-D₃] from its precursor, while those from mothers predominantly produced [³H]-24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃]. The identity of the radioactive metabolites produced from [³H]-25-OH-D₃ was established by periodate cleavage and comigration with synthetic 1α,25-(OH)₂-D₃ or 24,25-(OH)₂-D₃ on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)₂-D₃ synthesis in pregnancy.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

Viomycin resistance: alterations of either ribosomal subunit affect the binding of the antibiotic to the pair subunit and the entire ribosome becomes resistant to the drug.

Subcellular localization of $\text{[}{}^3\text{H]1α,24(R)-dihydroxyvitamin}$ D₃ and $\text{[}{}^3\text{H]1α,24(S)-dihydroxyvitamin}$ D₃ in rat intestinal mucosa was investigated in comparison with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃. The 24(R) and 24(S) isomers of 1α,24-dihydroxyvitamin D₃ were gradually transformed to 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃, and the plasma concentrations of these metabolites were 10.30 and 1.36 pmol/ml, respectively. The major portions of the administered compounds distributed in the nuclear fraction of the intestinal mucosa remained unchanged, and the amounts of 1α,24(R)-dihydroxyvitamin D₃ and 1α,24(S)-dihydroxyvitamin D₃ were 4.25 and 0.306 pmol/g intestinal mucosa, respectively. No detectable amount of the metabolites, 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃ were found in the same nuclear fractions. In the case with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃, however, the compound was rapidly metabolized to 1α,25-dihydroxyvitamin D₃.The metabolite, 1α,25-dihydroxyvitamin D₃, was seen in the nuclear fraction of the intestinal mucosa at a concentration of 2.44 pmol/g intestinal mucosa.