Equine rhabdomyolysis.

A 1.5-year-old Quarter Horse gelding with a history of chronic nasal discharge and leukocytosis presented with signs of increased lethargy and muscular pain. The horse quickly became recumbent and unable to rise and was euthanized due to a poor prognosis. At necropsy, severe bilateral guttural pouch empyema was observed, as well as numerous well-demarcated areas of pallor within the skeletal muscles of all major muscle groups. Polymerase chain reaction testing of the guttural pouch exudate confirmed an infection with Streptococcus equi subsp. equi, and an S. equi-associated immune-mediated rhabdomyolysis was initially considered to be the most likely diagnosis. This report briefly discusses the various etiologies that should be considered in cases of equine myopathy, and it demonstrates the complexity of these poorly understood muscular disorders.

Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals.

Presynaptic nicotinic acetylcholine receptors likely play a modulatory role in the nerve terminal. Using laser-scanning confocal microscopy, we have characterized physiological responses obtained on activation of presynaptic nicotinic receptors by measuring calcium changes in individual nerve terminals (synaptosomes) isolated from the rat corpus striatum. Nicotine (500 nM) induced Ca(2+) changes in a subset (10-25%) of synaptosomes. The Ca(2+) responses were dependent on extracellular Ca(2+) and desensitized very slowly (several minutes) on prolonged exposure to agonist. The nicotine-induced Ca(2+) responses were dose-dependent and were completely blocked by dihydro-beta-erythroidine (5 microM), differentially affected by mecamylamine (10 microM) and alpha-conotoxin MII (100 nM), and not affected by alpha-bungarotoxin (500 nM). Immunocytochemical studies using well-characterized monoclonal antibodies revealed the presence of the alpha4 and alpha3/alpha5 nicotinic subunits. The nicotine-induced responses were unaffected by prior depolarization or by a mixture of Ca(2+) channel toxins including omega-conotoxin MVIIC (500 nM), omega-conotoxin GVIA (500 nM) and agatoxin TK (200 nM). Our results indicate that nicotinic receptors present on striatal nerve terminals induce Ca(2+) entry largely without involving voltage-gated Ca(2+) channels, most likely by direct permeation via the receptor channel itself. In addition, at least two subpopulations of presynaptic nicotinic receptors reside on separate terminals in the striatum, suggesting distinct modulatory roles.

Functional IP3- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108-15 (rodent neuroblastoma x glioma hybrid) cells.

Presynaptic varicosities of the model neuronal cell line NG108-15, a cholinergic neuroblastoma cell x glioma cell hybrid capable of innervating striated myotubes, were examined for the presence of inositol 1,4,5-trisphosphate (IP3)-sensitive and Ca2+-activated (ryanodine-sensitive) Ca2+ stores using confocal microscopic imaging of Ca2+-sensitive fluorescent dye loaded into the cells. Initial demonstration of the presence of IP3 receptors and ryanodine receptors in the NG108-15 varicosities was obtained using immunocytochemistry. Treatment of NG108-15 cells with bradykinin (0.1 microM), whose receptor is linked to IP3 generation, and separately, caffeine (10 mM), an activator of endoplasmic reticulum ryanodine receptors, resulted in substantial increases in [Ca2+]i in the varicosities. K+-evoked changes in [Ca2+]i in the varicosities were reduced (52 %) after emptying the ryanodine-sensitive Ca2+ store using caffeine (10 mM), but were not affected by prior depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 microM). Bradykinin-induced changes in [Ca2+]i were abolished following depletion of the IP3-sensitive Ca2+ store using thapsigargin (1 microM) and were reduced (72 %) by prior emptying of the ryanodine-sensitive Ca2+ store with caffeine (10 mM). The same results were obtained when the varicosities of the NG108-15 cells had formed synaptic junctions with co-cultured rat hindlimb myotubes. Taken together, the results suggest that, in the varicosities, activation of the IP3 pathway evoked the release of Ca2+ from the IP3-sensitive store, which, in turn, secondarily induced the release of Ca2+ from the ryanodine-sensitive store via Ca2+-induced Ca2+ release, and that depolarization-induced Ca2+ entry evoked Ca2+-induced Ca2+ release only from the ryanodine-sensitive store. Thus, functional internal Ca2+ stores are inherent components of presynaptic varicosities in this neural cell line.

Inhibitors of serine/threonine-specific protein phosphatases stimulate transcription by the Ah receptor/Arnt dimer by affecting a step subsequent to XRE binding.

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a dimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/ Arnt dimer can be decreased by treatments causing the down-regulation of protein kinase C and decreasing the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid caused two- to threefold increases in TCDD-dependent transcription at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubled TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increased TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of okadaic acid or cyclosporin A which substantially stimulated TCDD-dependent transcription. Neither of the inhibitors significantly increased the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increased the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription.

The DNA binding of purified Ah receptor heterodimer is regulated by redox conditions.

The Ah receptor from rat liver has been purified, using a specific oligonucleotide affinity column, in order to characterize the components of the receptor and to investigate features that modulate its DNA-binding activity. The purified DNA-binding form of rat Ah receptor contains three major components, with estimated molecular masses of 108, 98, and 96 kDa. Antibodies to two peptides from the mouse Ah receptor bind the 108-kDa protein, but not the 98-kDa protein, and bind weakly at the position of the 96-kDa protein. The sequences of four peptides from samples containing both the 96- and 98-kDa proteins are all highly similar to segments of the human Ah receptor nuclear translocator (Arnt) protein. Antibodies to a peptide from the human Arnt protein bind the 96- and 98-kDa proteins, but not the 108-kDa protein. These data show that the Ah receptor itself and two forms of the Arnt protein are the major components of the purified DNA-binding form of receptor. In gel shift assays the purified receptor forms two specifically bound complexes with a xenobiotic responsive element (XRE), which may correspond to Ah receptor heterodimers with either of the two forms of Arnt protein. The DNA binding of the purified heterodimer is substantially decreased under oxidizing conditions. Oxidation inhibits receptor DNA binding without greatly altering the size of the purified heterodimer. This sediments at 5.9S in its reduced form and at 6.5S in its oxidized form. Dithiothreitol restores the XRE binding of oxidized receptor, with similar effects on both of the receptor-XRE complexes. In the presence of nuclear extract, reduced thioredoxin also restores the XRE binding of oxidized receptor.

Galactose-1-phosphatase in rat brain.

A prominent galactose-1-phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl-Sephacel, hydroxylapatite, and Sephacryl S-300 columns. The galactose-1-phosphatase was separated from alkaline phosphatase, and from two forms of glucose-1-phosphatase. The three columns gave a 10-fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose-1-phosphate was the best substrate for the purified enzyme, followed by glucose-1-phosphate, which was hydrolyzed 40% as rapidly as galactose-1-phosphate. Galactose-1-phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose-1-phosphate hydrolysis. Mg2+ was required for activity, and supported half-maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L-homoarginine, and L-phenylalanine stimulated enzyme activity. Galactose-1-phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.1S. These values were used to calculate a molecular weight of 50,200 and a frictional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose-1-phosphate concentrations in patients with galactosemia.

ATP promotes the appearance of two DNA-binding forms of the Ah receptor.

Following the binding of a specific ligand such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, mild heating has previously been shown to convert cytosolic aryl hydrocarbon (Ah) receptor to two DNA-binding forms: peak I, which appears to be an Ah receptor monomer; and peak II, which is larger and resembles nuclear Ah receptor. In rat liver cytosol pretreated with charcoal-dextran, and heated briefly at 22 degrees C, the addition of a physiological (3 mM) concentration of ATP substantially increases the formation of both peak I and peak II receptor. On more extended incubation in the presence of ATP most of the Ah receptor converts to the tighter binding peak II form. The ATP analog 5'-adenylylmethylene diphosphonate (AMPPCP) stimulates the appearance of both DNA-binding forms as effectively as ATP does. In cytosol separated from low molecular weight components by gel filtration prior to incubation, ATP substantially stimulates the appearance of peak II receptor. ATP also increases the amount of peak II receptor formed when peak I receptor is incubated with unlabeled charcoal-treated cytosol. Thus, ATP promotes both the release of Ah receptor monomer from the 9 S complex which cannot bind DNA and the subsequent conversion of that monomer to a form similar or identical to nuclear Ah receptor.

Nucleoside triphosphates promote the transformation of Ah receptor to its DNA-binding form.

When it is bound to a specific ligand such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, mild heating can convert the Ah (aryl hydrocarbon) receptor into a form capable of binding DNA. We found that physiological (1-3 mM) levels of ATP substantially increased the transformation of the receptor to its DNA-binding form. GTP, UTP and CTP had similar effects. ADP also promoted this transformation, but was less effective than ATP at low concentrations. Pyrophosphate too promoted transformation, but AMP had little effect. The process did not require nucleotide hydrolysis, since non-hydrolysable analogues of ATP such as adenosine 5'-[beta gamma-imido]triphosphate were nearly as effective as ATP itself. Inhibitors of ATP-stimulated proteases did not significantly affect the ability of ATP to promote receptor transformation, which suggests that the effect of ATP was not mediated by these proteases.

High-performance tryptic mapping of recombinant bovine somatotropin.

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.

pH-induced conformational states of bovine growth hormone.

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation and characterization of 7 and 9 S forms of rat liver aryl hydrocarbon receptor.

Two forms of rat liver aryl hydrocarbon receptor were separated by chromatography on DEAE-cellulose in the presence of molybdate. After labeling for 2 h at 0 degrees C, the receptor separated on the DEAE column into a flow-through peak (peak I) and a peak eluting at 80 mM KCl (peak II). It had been reported previously that exposure to high salt in the presence of molybdate caused the appearance of both 9 and 5-6 S receptor forms. After confirming this, I examined the relationship of the peak I and peak II receptors to these receptor forms. In high salt buffer containing molybdate, the peak I receptor sedimented in the 5-6 S region and the peak II receptor at 9 S. High salt buffer lacking molybdate converted both peak I and peak II receptors to forms sedimenting in the 5-6 S region. In low salt buffer containing molybdate, the peak I receptor sedimented at slightly more than 7 S and the peak II receptor at 9-10 S. Thus, the peak II receptor could be stabilized by molybdate as a 9 S form, and the peak I receptor was converted by high salt from a 7 to a 5-6 S form, despite the presence of molybdate. Most of the peak I receptor bound to a DNA-cellulose column and was eluted by high salt. The peak II receptor showed very little DNA binding.

Characterization of purified avian 90,000-Da heat shock protein.

The so-called 90,000-Da heat shock protein (hsp90) from chicken liver has been purified and physically characterized in the presence of high levels of the serine phosphatase inhibitor fluoride. The protein is an elongated dimer with a molecular weight of 160,000 and a frictional ratio of 1.6. On two-dimensional electrophoresis it exhibits several isoelectric forms lying between pH 5.1 and 5.8. It contains an average of 5.8 mol of covalently bound phosphate per dimer and is thus extensively phosphorylated. Analysis of the ultraviolet spectrum showed the purified protein to be free of nucleotide-containing components. Molybdate has been shown to stabilize complexes between the 90,000-Da heat shock protein and steroid receptors. However, molybdate has no effect on the sedimentation of the purified heat shock protein. Proteins structurally related to hsp90 have been reported to penetrate the endoplasmic reticulum. However, when purified hsp90 was tested using the partition method of Bordier, which distinguishes hydrophilic and lipophilic proteins, it partitioned totally into the aqueous phase.

The kinetics of bovine growth hormone folding are consistent with a framework model.

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.

Identification of the 90 kDa substrate of rat liver type II casein kinase with the heat shock protein which binds steroid receptors.

It was recently reported that the type II casein kinase of rat liver cytosol co-purified with a major 90 kDa substrate when subjected to gel filtration at low ionic strength. The identity of the 90 kDa substrate was unknown. We have verified this report and have shown that the 90 kDa substrate is recognized by a monoclonal antibody prepared against the 90 kDa heat shock protein. This ubiquitous phosphoprotein is known to increase in abundance in cells subjected to heat stress and has been shown to complex steroid receptors and certain retrovirus tyrosine kinases.

Reoxidation of reduced bovine growth hormone from a stable secondary structure.

In order to determine solution conditions appropriate for reoxidizing reduced bovine growth hormone (bGH), we have examined the possibility of using a particular denaturant concentration to poise the secondary and tertiary structure of the reduced protein in a stable, nativelike state. It was envisioned that the structure of the reduced molecule would differ from that of the final oxidized molecule solely by the absence of disulfide bonds. Dilution of concentrated samples of reduced and unfolded protein from 6.0 M guanidine into 4.5 M urea followed by air oxidation indicated it was possible to induce refolding and reoxidation to an oxidized monomeric species in high yield (approximately 90%). The choice of solution conditions was based on comparison of urea equilibrium denaturation data for native oxidized protein to those for completely reduced protein and to protein in which sulfhydryl groups had been either partially or completely reduced and subjected to modification with iodoacetamide or methyl methanethiolsulfonate. The denaturation behavior of these species supports the existence of equilibrium folding intermediates for bovine growth hormone and demonstrates that chemical modification of the protein is capable of inducing differences in the denaturation behavior of these intermediates. The changes in the protein absorption spectrum and helix-related circular dichroism signal, along with direct titration of protein sulfhydryl groups, indicated that the refolding/reoxidation of bGH is a multistate process. The ordered nature of the kinetic changes in these probes during reoxidation indicates that disulfide formation is a sequential process, with little mispairing in 4.5 M urea, and that it proceeds through one or more obligatory kinetic folding events. The equilibrium denaturation behavior of the oxidized molecule and the various chemically modified forms, together with the reoxidation data, indicated that the protein maintains a high degree of secondary structure without intrachain disulfide bonds. The formation of these disulfide bonds is a discrete process which occurs after a framework of protein secondary structure is established.

Polypeptide components of two 8 S forms of chicken oviduct progesterone receptor.

Two 8 S forms of progesterone receptor from the chicken oviduct were purified to near homogeneity and analyzed for peptide composition by gel electrophoresis. Form I contains two major peptides with molecular weights of 90,000 and 75,000. Form II also contains two major peptides with molecular weights of 90,000 and 110,000. In glycerol gradients containing molybdate, the 90,000, 110,000, and 75,000 molecular weight peptides co-sediment with the [3H]progesterone peak at 8 S. In high salt gradients lacking molybdate, the [3H]progesterone peak co-sediments with the 110,000 and 75,000 molecular weight peptides at 4 S, while the 90,000 molecular weight peptide sediments at 6-7 S. On photoactivation, the synthetic progestin R5020 binds covalently to the 110,000 and 75,000 molecular weight peptides. Thus, both 8 S forms of progesterone receptor contain 90,000 molecular weight peptides which do not bind progesterone, and each 8 S form contains a separate form of progesterone-binding peptide. When receptor is purified from oviduct minces incubated with [32P]orthophosphate, autoradiography indicates the presence of 32P in the 90,000 and 110,000 molecular weight peptides and in a peptide which appears to be slightly larger than 75,000 and may be a more highly phosphorylated fraction of the 75,000 molecular weight peptide. Thus, three separate peptides have been identified as components of the 8 S form of progesterone receptor, and all three appear to exist as phosphoproteins.

The avian progesterone receptor: isolation and characterization of phosphorylated forms.

Methods have been developed for isolation of the avian progesterone receptor in the nontransformed, molybdate-stabilized state. The final step in this procedure. DEAE-Sephadex chromatography, resolves the receptor into two forms, components I and II. Analysis of these components by polyacrylamide gel electrophoresis under denaturing conditions shows that both contain a peptide with Mr = 90,000. These peptides contain phosphorylated serine residues, as shown by [32P]orthophosphate incorporation studies. When the cytosol receptor is treated with alkaline phosphatase, its steroid binding capacity is abolished. These studies show that the nontransformed progesterone receptor is a phosphoprotein and indicate that receptor phosphorylation may be important to the maintenance of steroid binding capacity.

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria.

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26 000; MgGTP + AMP in equilibrium MgGDP + ADP) has been attained by treatment of the enzyme with N-bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (lambda exc 295 nm) were recorded over the pH range 1.9-12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M-1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N-bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride. In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the lambda max of emission shifts from 333-334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to tyrosinate.

Phosphorylation in vivo of chicken oviduct progesterone receptor.

Progesterone receptor from an oviduct tissue mince incubated with (32P)orthophosphate was purified using affinity chromatography, gel filtration, and DEAE-Sephadex chromatography. Two receptor peaks were eluted from the DEAE column. Peak I contained one major protein band with a molecular weight of 90,000 and a 32P band at 90,000. Peak II also had a major 90,000-dalton protein band and a 32P band at the same position. In addition, peak II contained a major protein band at 104,000 daltons and a 32P band which appeared to be slightly larger. Peaks I and II from chickens injected with (32P)orthophosphate showed the same pattern of labeling. The 90,000-molecular weight proteins from peaks I and II and the 32P-labeled component of II with Mr greater than 104,000 all contained phosphoserine. On two-dimensional gels, the 90,000-dalton proteins from peaks I and II were indistinguishable. Both contained labeled phosphate. Two-dimensional gels separated the 104,000-dalton peptide from the slightly larger 32P-labeled protein which traveled close to it on a one-dimensional gel. The relationships of the 104,000-dalton protein and this phosphoprotein to the progesterone receptor are not known. The 90,000-dalton receptor form common to both peaks is a phosphoprotein.

Purification of "nontransformed" avian progesterone receptor and preliminary characterization.

The nontransformed (molybdate-stabilized) avian oviduct progesterone receptor has been purified to near homogeneity by a simple four-step procedure: ammonium sulfate fractionation, affinity chromatography, gel filtration, and DEAE-Sephadex A-25 chromatography. The affinity resin (deoxycorticosterone-agarose) is very resistant to chemical breakdown and enzymatic destruction and can be used repeatedly. The material obtained after gel filtration was separated into two receptor components by DEAE-chromatography. The two components, termed I and II, are obtained in about 18% yield and are purified by about 6000-fold (I) and 4000-fold (II). They are highly purified as only a single major Coomassie blue-stained polypeptide of about Mr = 90,000 in both components I and II is seen on sodium dodecyl sulfate-gel electrophoresis plus a second band of Mr = 104,000 which is seen only in component II. Both purified receptor components have sedimentation coefficients of 8 S in glycerol gradients containing molybdate (10 mM). In the absence of molybdate, however, both receptor forms have sedimentation coefficients of about 4 S in gradients containing 300 mM KCl. Therefore, stabilization of the 8 S form by molybdate is still evident in highly purified receptor preparations. The Stokes radii, binding specificity, and steroid dissociation rate of purified receptor are similar to those found in crude cytosol. These results confirm our previous observation on the existence of two 8 S forms of the progesterone receptor and show that these molybdate-stabilized forms remain intact throughout purification.