Stereospecific formation of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid from either (25R)- or (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid by rat liver homogenate

Studies of the stereochemistry of the intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, in the biosynthetic sequence between 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and cholic acid have been undertaken. (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-Trihydroxy-5 beta-cholestan-26-oic acid was incubated with rat liver homogenates. The reaction products were converted to p-bromophenacyl ester derivatives and the esters were analyzed by high-performance liquid chromatography. By comparison with authentic samples of two (24E)- and (24Z)-isomers of the alpha, beta-unsaturated acid and of four isomers at C-24 and C-25 of the beta-hydroxy acid, (24E)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid were found to be formed from either (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. No formation of the (24Z)-isomer of the trihydroxycholestenoic acid or the other three isomers of the tetrahydroxycholestanoic acid was detected. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.     

The lactam of alpha-guanidinoglutaric acid (1-amidino-2-pyrrolidone-5-carboxylic acid)

alpha-Guanidinoglutaric acid (alpha-GGA) has been reported to occur in the cerebral cortex after epileptic seizures. No physical characteristics of alpha-GGA have been given. A practical procedure for the preparation of alpha-GGA is reported here. alpha-GGA forms a lactam in aqueous solution at 80 degrees C. It is proposed to substitute this lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid (pAGlu), for pyroglutamic acid (pGlu) at the N-terminal position in neuropeptides to modify their biological characteristics. L(+)-Glutamic acid was reacted with S-methylisothiourea (I) at pH 10 in aqueous solution to form L(-)-alpha-guanidinoglutaric acid: mp 165-168 degrees C, [alpha]22D = -22.7 (C = 4, 2 M HCl). alpha-GGA reacted promptly with excess reagent to form a salt, S-methylisothiourea-alpha-guanidinoglutarate: mp 209-210 degrees C, [alpha]22D = -13.0 (C = 4, 2 M HCl). I was removed from the salt with aqueous picric acid, since I readily formed an insoluble picrate, S-methylisothiourea picrate (mp 225-228 degrees C). Alternatively, the salt was added to a cation exchange column, and the alpha-GGA was eluted with molar ammonium acetate buffer, pH 9.5. Its lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid, mp 248-249 degrees C, [alpha]22D = +2.1 (C = 4, 2 M HCl), formed a picrate (mp 196-199 degrees C).     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1alpha,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the previously well established main side chain modification pathway, is initiated by hydroxylation at C-24 of the side chain. The C-3 epimerization pathway, the newly discovered A-ring modification pathway, is initiated by epimerization of the hydroxyl group at C-3 of the A-ring. The end products of the metabolism of 1alpha,25(OH)2D3 through the C-24 oxidation and the C-3 epimerization pathways are calcitroic acid and 1alpha,25-dihydroxy-3-epi-vitamin-D3 respectively. During the past two decades, numerous noncalcemic analogs of 1alpha,25(OH)2D3 were synthesized. Several of the analogs have altered side chain structures and as a result some of these analogs have been shown to resist their metabolism through side chain modifications. For example, two of the analogs, namely, 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-D3] and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-D3], have been shown to resist their metabolism through the C-24 oxidation pathway. However, the possibility of the metabolism of these two analogs through the C-3 epimerization pathway has not been studied. Therefore, in our present study, we investigated the metabolism of these two analogs in rat osteosarcoma cells (UMR 106) which are known to express the C-3 epimerization pathway. The results of our study indicate that both analogs [1alpha,25(OH)2-16-ene-23-yne-D3 and 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3] are metabolized through the C-3 epimerization pathway in UMR 106 cells. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-D3 [1alpha,25(OH)2-16-ene-23-yne-3-epi-D3] was confirmed by GC/MS analysis and its comigration with synthetic 1alpha,25(OH)2-16-ene-23-yne-3-epi-D3 on both straight and reverse-phase HPLC systems. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-3-epi-D3] was confirmed by GC/MS and 1H NMR analysis. Thus, we indicate that vitamin D analogs which resist their metabolism through the C-24 oxidation pathway, have the potential to be metabolized through the C-3 epimerization pathway. In our present study, we also noted that the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 is about 10 times greater than the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-D3. Thus, we indicate for the first time that certain structural modifications of the side chain such as 20-epi modification can alter significantly the rate of C-3 epimerization of vitamin D compounds.     

Structural and biosynthetic studies of a principal bile alcohol, 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24,25-pentol, in human urine

The stereochemistry at C-24 and C-25 of 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24 ,25-pentol, a principal bile alcohol in human urine, and its biosynthesis are studied. Four stereoisomers of the C(26)-24,25-pentols were synthesized by reduction with LiAlH(4) of the corresponding epoxides prepared from (24S)- or (24R)-27-nor-5beta-cholest-25-ene-3alpha, 7alpha,12alpha,24-tetrol. The stereochemistries at C-25 were deduced by comparison of the C(26)-24,25-pentols with the oxidation products of (24Z)-27-nor-5beta-cholest-24-ene-3alpha,7alpha, 12alpha-triol with osmium tetraoxide. On the basis of this assignment, the principal bile alcohol excreted into human and rat urine was determined to be (24S,25R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha,24,25-pentol, accompanied by a lesser amount of (24R, 25R)-isomer. To elucidate the biosynthesis of the C(26)-24,25-pentol, a putative intermediate, 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestan-24-one, derived from 3alpha,7alpha, 12alpha-trihydroxy-24-oxo-5beta-cholestanoic acid by decarboxylation during the side-chain oxidation of 3alpha,7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid, was incubated with rat liver homogenates. The 24-oxo-bile alcohol could be efficiently reduced to yield mainly (24R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha,24-tetrol. If a 25R-hydroxylation of the latter steroid occurs, it should lead to formation of (24S,25R)-C(26)-24,25-pentol. Now it has appeared that a major bile alcohol excreted into human urine is (24S,25R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha, 24, 25-pentol, which might be derived from 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestan-24-one via (24R)-27-nor-5beta-cholestane-3alpha, 7alpha,12alpha,24-tetrol.     

New bile alcohols--synthesis of 5beta-cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24 (14C)-triol.

5beta-Cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24(14-C)-triol were synthesized from 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid). Chenodeoxycholic acid was converted to the diformoxy derivative (II) using formic acid. Reaction of II with thionyl chloride yielded the acid chloride which was treated with diazomethane (CH-2-N-2 or 14-CH-2-N-2) to produce 3alpha, 7alpha-diformoxy-24-oxo-25-diazo-25-homocholane (III, A or B). 25-Homochenodeoxycholic acid (IV, A or B) was formed from III by means of the Wolff rearrangement of the Arndt-Eistert synthesis. The methyl ester of V (A or B) was treated with methyl magnesium iodidi in ether to provide the desired triol, VI (A and B). The triol was identified by mass spectrometry and elemental analysis and was characterized by thin-layer and gas-liquid chromatography. The 3alpha, 7alpha, 25-triol is of possible significance as an intermediate in the pathway of bile acid formation from cholesterol.     

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.     

Configurational assignment of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol excreted by patients with cerebrotendinous xanthomatosis (a circular dichroism study).

The absolute configuration of the C27 pentahydroxy bile alcohol present in bile and feces of two patients with cerebrotendinous xanthomatosis (CTX) was determined by circular dichroism (CD) spectroscopy. Under anhydrous conditions CD spectra of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol in the presence of Eu (fod) 3[tris (1, 1, 1, 2, 2, 3, 3-hepta fluoro-7, 7-dimethyl-octane-4, 6-dionato) europium (III)] exhibited a large induced split Cotton effect at ca. 310 nm. From the induced circular dichroism of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol with Eu(fod) 3 it was concluded that the CTX bile alcohol has the 1, 3 glycol structure with carbon 23 having the R configuration. This information will be useful in elucidating a structural mechanism for the conversion of 5 beta-cholestranepentols into bile acids in man and rat.     

Identification of pentahydroxy bile alcohols in cerebrotendinous xanthomatosis: characterization of 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 23xi, 25-pentol.

This paper describes studies dealing with the nature of the C27 pentahydroxy bile alcohols present in the bile and feces of two patients with cerebrotendinous xanthomatosis (CTX). The presence of a bile alcohol having the structure 5beta-cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol was confirmed by separation of the two 24-hydroxy epimers of 5beta-cholestane-3alpha,7alpha,12alpha,24,25-pentol and characterization of the dpimers by gas-liquid chromatography and infrared and mass spectrometry. Tentative assignment of the 24alpha and 24beta configuration was made on the basis of molecular rotation differences. A second major bile alcohol excreted by the CTX subjects was 5beta-cholestane-3alpha,7alpha,12alpha,23xi,25-pentol. Its structure was determined by infrared spectrometry, proton magnetic resonance spectrometry, and mass spectrometry because a reference compound was not available.     

Synthesis of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 245, 25-pentol.

This paper describes syntheses of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol which give higher yields than previously published methods. In addition, 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol was synthesized by a different procedure, namely via performic acid oxidation of the correspinding unsaturated triol, which gave a lower yield but avoided the formation of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol, which normally tends to contaminate the final product. Structures were confirmed by gas-liquid chromatography, infrared-, proton magnetic resonance- and mass spectrometry, 5beta-Cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol were required for in vivo and in vitro studies of the (hypothetical) 25-hydroxylation pathway of cholic acid biosynthesis.     

Absolute configuration at carbon 23 of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol excreted by patients with cerebrotendinous xanthomatosis

Absolute configuration at C-23 of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, one of the bile alcohols isolated from the patients with cerebrotendinous xanthomatosis, was unequivocally determined as 23S by conversion of a key intermediate, (23S)-5 beta-cholest-25-ene-3 alpha,7 alpha,12 alpha,23-tetrol to either the bile alcohol of known absolute configuration, (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol, or the naturally occurring 23,25-pentol.     

Biophysical studies on molecular mechanism of abortifacient action of prostaglandins. VI. Conformation energy calculation on PGE2, PGF2 alpha and 15-(s)-methyl PGF2 alpha

This paper reports the conformation energy (CE) calculations on PGE2, PGE2 alpha and 15-(s)-methyl PGE2 alpha on the basis of empirical potential energy functions for the simultaneous rotations around C7-C8 (psi), C12-C13 (theta) and C14-C15 (phi) bonds. The variation of the minimum conformation energy E for each isoenergy map in the psi theta plane with respect to phi gives two minima around 90 degrees and 240 degrees in PGE2, 60 degrees and 245 degrees in PGF2 alpha, and 60 degrees and 150 degrees in 15-(s)-methyl PGF2 alpha. The latter two forms also have a small dip at 270 degrees. The pattern of allowed low energy conformations of PGF2 alpha and 15-(s)-methyl PGF2 alpha is quite similar and is characterized by the existence of six low energy regions.     

Synthesis and structure of 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol isolated from the urine and feces of a patient with sitosterolemia and xanthomatosis

The urine and feces of a patient with the rare inherited lipid storage disease, sitosterolemia and xanthomatosis, were analyzed. Substantial quantities of C26-bile alcohol, 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol along with 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24R,25-pentol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol were found. The structure of the C26-bile alcohol was confirmed by direct comparison (gas-liquid chromatography-mass spectrometry and thin-layer chromatography) with a standard sample synthesized from cholic acid. The configurational assignment at C-24 was determined by lanthanide-induced circular dichroism Cotton effect measurements. The increased excretion of these C26- and C27-bile alcohols suggests an abnormality of bile acid biosynthesis in this disease.     

Differences in local conformation around cysteine residues in alpha alpha, alpha beta, and beta beta rabbit skeletal tropomyosin

The ability of 5,5'-dithiobis-2-nitrobenzoate (Nbs2) to form a disulfide crosslink between the Cys-190s of the alpha alpha and alpha beta molecular components of rabbit skeletal tropomyosin (Tm) and the Cys-36s and Cys-190s of purified beta beta was studied in separate experiments, as a function of urea concentration in 0.5 M NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 15 degrees C. In the absence of urea, complete reaction of the Cys-190s of Tm with Nbs2 as well as with 2- and 4-pyridine disulfide quantitatively produced two crosslinked species, alpha-alpha and alpha-beta, in a 60/40 ratio, respectively, visualized as bands on sodium dodecyl sulfate-polyacrylamide gels; similar reactions of beta beta produced both doubly (at Cys-36 and Cys-190) and singly crosslinked species (at Cys-190 as identified by amino acid analysis of separated tryptic peptides). In the presence of 4 M urea where the chains were unfolded and separated, only Nbs-blocked uncrosslinked species were obtained after complete reaction with Nbs2. The loss of Nbs2-crosslinking at increasing [urea] showed that the relative stability of the Cys-containing regions of the three species of Tm, alpha alpha, alpha beta, and beta beta increases in the order Cys-36 of beta beta, Cys-190 of alpha beta, Cys-190 of alpha alpha.     

The soybean vegetative storage proteins VSP alpha and VSP beta are acid phosphatases active on polyphosphates.

The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP alpha and VSP beta) and heterodimers (VSP alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP alpha) or pH 5.0 (VSP alpha/beta and VSP beta) in 0.15 M sodium acetate buffer at 25 degrees C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70 degrees C for 10 min. These phosphatases can liberate Pi from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PPi, and short chain polyphosphates. VSP alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PPi, and polyphosphates most efficiently. Apparent Km and Vmax values at 25 degrees C and pH 5.0 were 42 microM and 2.0 mumol/min/mg, 150 microM and 4.2 mumol/min/mg, and 420 microM and 4.1 mumol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.     

Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.     

Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.     

Occurrence of both (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids in urine from an infant with Zellweger's syndrome

Urine from a patient with Zellweger's syndrome was examined for bile acids after fractionation into three groups according to mode of conjugation. 3 alpha,7 alpha,12 alpha-Trihydroxy-5 beta-cholestanoic acid was the predominant bile acid of the unconjugated and glycine-conjugated bile acid fractions. Smaller amounts of cholic acid and 1 beta-, 6 alpha-, 24-, and 26-hydroxylated derivatives of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid were found in both fractions in similar proportions. The bile acid spectrum of the taurine-conjugated bile acid fraction was different from those of the other two fractions in the occurrence of two new compounds as the major constituents. These compounds were tentatively identified as two epimers at C-23 of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestano-26,23-lactone, which were probably artifacts formed from the corresponding tetrahydroxycholestanoic acids during the procedures for extraction after hydrolysis. High-performance liquid chromatographic analysis revealed that 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid excreted into the urine as the unconjugated form consisted of a mixture of (25R)- and (25S)-isomers in the ratio of about 7:3.     

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.