PubMed Health⌕ Search

Biomedical subjects

B E Haley

Publications and source records attributed to B E Haley.

At least 19 recordsLinked to original sources
PubMed2000

Creatine kinase in human retinal pigment epithelium.

Retinal pigment epithelial ion transport activity, and consequent ATP consumption vary significantly as a function of photoreceptor activity. In a variety of cell types, ATP levels are maintained during high-energy usage by phosphocreatine hydrolysis, catalysed by the enzyme creatine kinase. The present work was designed to assess the importance of creatine kinase in retinal pigment epithelial cell metabolism. To this end, activity measurements, non-denaturing gel electrophoresis, Western blot analysis and immunohistochemistry were used to characterize creatine kinase in retinal pigment epithelium. Total creatine kinase activity in the retinal pigment epithelium is approximately 0.05 micromol ATP mg protein(-1) min(-1). The bulk of this activity was mediated by the B-CK isoform. However, by immunoblotting, non-denaturing gel electrophoresis and immunohistochemistry, the presence of the M-CK isoform of creatine kinase was also detected. The M-CK isoform was plasma membrane associated and predominately localized to the apical surface. Creatine kinase in the retinal pigment epithelium could function in a spatial energy shuttle that helps to sustain apical plasma membrane ion transport activity.

Blotting, Western↗
PubMed1999

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.

Adenosine Triphosphate↗
PubMed1999

Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase.

Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate-binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP. Without activating light, GTPgammaBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPgammaBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+-chelate affinity chromatography and reversed-phase HPLC, photolabeled peptides located within or near the phosphate-binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPgammaBP primarily modified a peptide near the middle of the GDH sequence, Asn135-Lys143 and Glu290-Lys295. However, ATPgammaBP modified a single peptide corresponding to the sequence Met411-Arg419 near the C-terminal domain. Using these results and the data from the previously identified base-binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three-dimensional schematic model structure derived from the bacterial enzyme.

Adenosine Diphosphate↗
PubMed1998

Abnormal properties of creatine kinase in Alzheimer's disease brain: correlation of reduced enzyme activity and active site photolabeling with aberrant cytosol-membrane partitioning.

The report shows that Alzheimer's disease (AD) brain creatine kinase (CK) is modified such that the nucleotide binding site of CK is blocked and that abnormal partitioning of CK between the soluble and pellet fractions occurs. First, CK activity was 86% decreased in AD brain homogenates in comparison to age-matched controls. Secondly, over a 23.5 fold greater 32P photoincorporation of [alpha 32P]8N3ATP was observed into CK of control vs. AD samples. Also, a 7.4-fold increase of enzyme induced 32P incorporation was observed in controls vs. AD samples by incubation with [gamma 32P]ATP. Thirdly, Western blot analysis showed that CK copy numbers in the AD homogenate were decreased by less than 14% in comparison to controls. However, analysis showed that control supernatant and pellet fractions contained 10.3 and 0.4 times the CK copy number found in the corresponding AD fractions. 32P incorporation by both photolabeling and enzyme catalyzed incorporation of radiolabel followed CK activity and not CK copy number. Further, [alpha 32P]ADP and [gamma 32P]ATP incorporated 32P into control brain and purified brain CK equally well, indicating that a mechanism different from gamma-phosphoryl transfer is involved in the enzymatic incorporation of radiolabel. Also, the level of abnormal partitioning of CK into AD brain pellet correlated with the decreased [32P]8N3GTP photolabeling and abnormal partitioning of beta-tubulin, a protein known to be aberrantly modified in the AD brain. This indicates that a common chemistry is affecting both CK and tubulin in AD.

Alzheimer Disease↗
PubMed1997

Identification of an adenine-nucleotide-binding site on interferon alpha2.

Using 32P-labeled 2-azidoadenosine 5'-triphosphate (2N3ATP) and 8-azidoadenosine 5'-triphosphate (8N3ATP), we have identified a site on human interferon alpha2 (IFN-alpha2) that binds adenine nucleotides. The results from saturation and competition experiments demonstrated the specificity of the nucleotide interaction. Half-maximal saturation of IFN-alpha2 was observed at 10 microM 2N3ATP or 35 microM 8N3ATP. ATP effectively decreased photoinsertion of both photoaffinity analogs of ATP. Photoinsertion of 8N3ATP was enhanced by MgCl2, independent of the ionic strength, and exhibited an optimum pH between 7.0 and 7.5. Immobilized-Al3+ affinity chromatography and HPLC were used to purify the modified peptides from IFN-alpha2 that had been photolabeled with 8N3ATP and digested with trypsin or chymotrypsin. Overlapping-sequence analysis localized the sites of photoinsertion to the region corresponding to Lys121-Tyr135 in the amino acid sequence of IFN-alpha2, which almost perfectly overlaps a nuclear-localization signal (R120KYFQRITLYLKEKKY135).

Adenine Nucleotides↗
PubMed1997

A comparison of changes in nucleotide-protein interactions in the striatal, hippocampus and paramedian cortex after cerebral ischemia and reperfusion: correlations to regional vulnerability.

[32P]Azido-purine analogs of ATP and GTP were used to detect changes in phosphorylation and nucleotide binding induced by ischemia and subsequent reperfusion in rat brain striatum, hippocampus and paramedian cortex (PM cortex) tissues. Major changes in phosphorylation were observed for a 130-kDa protein, tentatively identified as the Ca2+ transport ATPase, and calcium/calmodulin-dependent protein kinase II (CaM Kinase II) in all tissues. However, recovery of the phosphorylation of the 130-kDa protein occurred only in the PM cortex on reperfusion. A 200-300% increase in [32P]8N3ATP photoinsertions was observed in the striatum and hippocampus regions for a 43-kDa protein with an isoelectric point of 6.8. This protein was identified as glutamine synthetase (GS) and the increase in binding was found to be due to both increased copy number and activation by Mn2+. An increase in [32P]8N3GTP photoinsertion into a 55-kDa protein, identified as the beta-subunit of tubulin, was found only in the striatum and hippocampus. This indicates the depolymerization of microtubulin in these tissues. These changes correlate to the vulnerability of the striatum and hippocampus to ischemia-induced neuronal death.

Animals↗
PubMed1997

Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics: relevance to Alzheimer's disease.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second-order reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated beta-amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

Alzheimer Disease↗
PubMed1997

Mercury vapor inhalation inhibits binding of GTP to tubulin in rat brain: similarity to a molecular lesion in Alzheimer diseased brain.

Hg2+ interacts with brain tubulin and disassembles microtubules that maintain neurite structure. Since it is well known that Hg vapor (Hg0) is continuously released from "silver" amalgam tooth fillings and is absorbed into brain, rats were exposed to Hg0 4h/day for 0, 2, 7, 14 and 28 d at 250 or 300 micrograms Hg/m3 air, concentrations present in mouth air of some humans with many amalgam fillings. Average rat brain Hg concentrations increased significantly (11-47 fold) with duration of Hg0 exposure. By 14 d Hg0 exposure, photoaffinity labelling on the beta-subunit of the tubulin dimer with [alpha 32P] 8N3 GTP in brain homogenates was decreased 41-74%, upon analysis of SDS-PAGE autoradiograms. The identical neurochemical lesion of similar or greater magnitude is evident in Alzheimer brain homogenates from approximately 80% of patients, when compared to human age-matched neurological controls. Total tubulin protein levels remained relatively unchanged between Hg0 exposed rat brains and controls, and between Alzheimer brains and controls. Since the rate of tubulin polymerization is dependent upon binding of GTP to tubulin dimers, we conclude that chronic inhalation of low-level Hg0 can inhibit polymerization of brain tubulin essential for formation of microtubules.

Administration, Inhalation↗
PubMed1996

Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase.

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.

Adenine↗
PubMed1996

Novel unconventional binding site in the variable region of immunoglobulins.

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

Affinity Labels↗
PubMed1995

Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].

Adenine↗
PubMed1995

Identification of N-terminus peptide of human granulocyte/macrophage colony stimulating factor as the site of nucleotide interaction.

The interaction of nucleotides with recombinant human granulocyte/macrophage colony stimulating factor (rhGM-CSF) has been investigated. Utilizing nucleotide photoaffinity probes [gamma 32P]-8N3ATP and [beta 32P]-8N3Ap4A, an analog of alarmone, the specificity of interaction was demonstrated by saturation of photoinsertion by these analogs and protection of photoinsertion by these analogs in the presence of natural nucleotide. The site of photoinsertion was tentatively determined to be Ser9. The photolabeled cytokine has lost most of its biological activity in a cellular proliferation assay, indicating a possible physiological role for this interaction.

Adenosine Triphosphate↗
PubMed1994

Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Photoaffinity labeling of Rubisco activase with 2- and 8-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.

Adenosine Triphosphate↗
PubMed1994

Photoaffinity labeling of creatine kinase with 2-azido- and 8-azidoadenosine triphosphate: identification of two peptides from the ATP-binding domain.

Two different analogs of ATP, [gamma-32P]2N3ATP and and [gamma-32P]8N3ATP, were used to photoaffinity label the MM and BB isoforms of rabbit cytosolic creatine kinase. Evidence that photoinsertion was within the ATP-binding domain was as follows: (1) Assays for creatine phosphate production demonstrated that [gamma-32]2N3ATP and [gamma-32P]8N3ATP are substrates for creatine kinase. (2) Enzymatic activity was inhibited by photolabeling with either analog. (3) Saturation of photoinsertion was observed for both analogs. Half-maximal saturation was observed at 5 microM [gamma-32P]2N3ATP or 12 microM (gamma-32P]8N3ATP. (4) Photoinsertion of both probes could be decreased by micromolar levels of ATP. Immobilized Al3+ affinity chromatography and HPLC were used to isolate the peptides modified by these probes. Overlapping sequence analysis of the isolated peptides from the tryptic and chymotryptic digests of the photolabeled MM isoform revealed that [gamma-32P]8N3ATP photoinserted into the peptide region corresponding to Val279-Arg291, whereas [gamma-32P]2N3-ATP photoinserted into Val236-Lys241. The corresponding peptide (Ile279-Arg291 and Val236-Lys241) from the BB isoform were shown to be selectively modified. We conclude that amino acid residues within the peptide regions 236-241 and 279-291 of rabbit cytosolic creatine kinase are localized within the binding domain for the adenine moiety of ATP. The results also demonstrate the effectiveness and selectivity of Al3+ as the chelating agent in immobilized metal affinity chromatography for the isolation of photolabeled peptides as well as its potential to enhance retention of radiolabel during HPLC.

Adenosine Triphosphate↗
PubMed1994

Interaction of nucleotides with acidic fibroblast growth factor (FGF-1).

A wide variety of nucleotides are shown to bind to acidic fibroblast growth factor (aFGF) as demonstrated by their ability to (1) inhibit the heat-induced aggregation of the protein, (2) enhance the thermal stability of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfated naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding of mononucleotides, diadenosine compounds (ApnA), and inorganic polyphosphates to aFGF is enhanced as the degree of phosphorylation of these anions is increased with the presence of the base reducing the apparent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N3-ATP, 2N3-ATP, 8N3-GTP, and 8N3-Ap4A) were employed to covalently label the aFGF nucleotide binding site. In general, Kd's in the low micromolar range are observed. Protection against 90% displacement is observed at several hundred micromolar nucleotide concentration. Using 8N3-ATP as a prototypic reagent, photolabeled aFGF was proteolyzed with trypsin and chymotrypsin and labeled peptides were isolated and sequenced resulting in the identification of 10 possible labeled amino acids (Y8, G20, H21, T61, K112, K113, S116, R119, R122, H124). On the basis of the crystal structure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptive polyanion binding site. On residue (T61) is more distally located but still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity chromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for heparin and a reduced ability to stimulate mitogenesis even in the presence of polyanions such as heparin. In conclusion, nucleotides bind apparently nonspecifically to the polyanion binding site of aFGF but nevertheless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and the potential biological significance of these results in terms of potential natural ligands of aFGF are also discussed but not resolved.

Affinity Labels↗
PubMed1994

Identification of peptides within the base binding domains of the GTP- and ATP-specific binding sites of tubulin.

Using gamma-32P-labeled 8-azidopurine nucleotide photoaffinity probes of GTP and ATP, the respective purine ring binding domain peptides of tubulin have been identified. First, the location of the GTP-specific binding site was shown to be on the beta-subunit, whereas the major ATP-specific binding site was on the alpha-subunit. Using a combination of anion-exclusion and immobilized Al3+ column chromatography, the respective photolabeled tryptic peptides of both nucleotide binding sites were isolated, further purified by reverse phase high performance liquid chromatography (HPLC) and sequenced. Chymotryptic peptides were also generated for the GTP binding site. High retention of the photoinserted radiolabel was observed with many of the peptides on reverse phase HPLC at low flow rates. The stability of the photoinserted radiolabel to HPLC varied with different peptides. However, certain peptides were easily distinguished as being within the base binding domains of the GTP and ATP binding sites of tubulin. Two beta-tubulin peptides containing the majority of photoinserted [gamma-32P]8-azidoguanosine 5'-triphosphate corresponded to N-terminal beta-tubulin amino acid residues 3EIVHIQAGQCGNQIGAK19 and 20FWEVISDEHGIDPTGS35. The peptide containing the majority of photoinserted [gamma-32P]8-azidoadenosine 5'-triphosphate corresponded to the C-terminal alpha-tubulin sequence 431DYEEVGVDSVEGEGEEEGEE450.

Adenosine Triphosphate↗
PubMed1994

Photoaffinity labeling of rat liver microsomal steroid 5 alpha-reductase by 2-azido-NADP+.

Preincubation of female rat liver microsomal preparations with [2'-32P]2N3-NADP+ followed by photolysis with UV light (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32P into at least 3 major protein bands in the molecular weight range of 14-97 Kd. Labeling of a 26 kD band, the apparent molecular weight of 5 alpha-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP+ (100 microM) completely inhibited the incorporation of [2'-32P]2N3-NADP+ and preserved the enzyme activity, whereas excess NAD+ (100 microM) failed to protect 5 alpha-reductase (5 alpha R) activity. Similar results were obtained with the detergent-solubilized form of 5 alpha R. Polyethylene glycol (PEG) fractionation of detergent-solubilized preparations of 5 alpha R showed that all the 5 alpha R activity could be recovered in the 6.5% pellet with a 3-4-fold increase in the specific activity. photolysis of this fraction with [2'-32P]2N3-NADP+ resulted in approximately 2-fold increase in 32P labeling of the 5 alpha R band. Increasing photolysis time and concentration of the [2'-32P]2N3-NADP+ indicated that the half-life for photoincorporation and the apparent Kd were 1.0 min and 2 microM, respectively. These results suggest that 2N3-NADP+ is an effective probe of the NADP(H) binding site of 5 alpha R, and is a useful marker during purification of the enzyme.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗