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At least 19 recordsLinked to original sources
PubMed1973

The mechanism of photoaffinity labeling.

Photoaffinity labeling is a recently introduced method for covalently binding chemical tags to the active sites of protein molecules, which is potentially capable of very great specificities of labeling. A labeling reagent is used that is converted by photolysis to an extremely reactive intermediate. According to the expected mechanism, the reagent molecules that are specifically and reversibly bound to the active site at the instant of photolysis react irreversibly in the site before they can dissociate from the site. In two such reagent-protein systems studied in this paper, however, it is shown that, while by the usual criteria photoaffinity labeling appears to have occurred, the expected mechanism in fact does not hold. This was discovered in experiments with scavengers present in the mixtures that were photolyzed. The general properties of, and criteria for, photoaffinity labeling reactions are discussed in the light of these findings.

Acetylcholinesterase↗
PubMed1986

Identification of the D2-dopamine receptor binding subunit in several mammalian tissues and species by photoaffinity labeling.

Photoaffinity labeling of the D2-dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2-dopaminergic antagonist probe [125I]N-(p-azidophenethyl)spiperone ([125I]N3-NAPS). In tissues containing D2-receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr = 94,000. Specificity of the labeling is typically D2-dopaminergic in character. The covalent labeling is blocked by (+)-butaclamol but not by the inactive (-)isomer. Agonists block incorporation with the order of potency: N-n-propylnorapomorphine greater than apomorphine greater than dopamine. The D2-selective antagonist spiperone blocks labeling of the Mr = 94,000 peptide whereas the D1-selective antagonist SCH-23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D2-dopamine receptor resides on a Mr = 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower Mr (32-38,000) are labeled at the expense of the Mr = 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower Mr receptor fragments might be the result of a metal-dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D2-receptors, [125I]N3-NAPS specifically labels a major peptide of Mr approximately equal to 120,000 in addition to the Mr = 94,000 peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1979

Bile salt binding to serum components. Taurocholate incorporation into high-density lipoprotein revealed by photoaffinity labelling.

1. Photoaffinity labelling of human serum albumin with the sodium salts of (3 beta-azido-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid and (11 zeta-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid resulted, in each case, in a considerable covalent incorporation of radioactivity into the protein. 2. Photoaffinity labelling of whole serum, obtained from fasting test persons, revealed with all three photolabile derivatives of taurocholate at the physiological concentration of 2.1 microM the incorporation of radioactivity not only into albumin but also into high-density lipoprotein, as demonstrated by density gradient centrifugation and by immunological characterization. 3. The bulk of radioactivity incorporated into high-density lipoprotein by photoaffinity labelling of whole serum was found to have been associated with the lipids. Only 10-20% of the label was covalently bound to apolipoproteins, predominantly to the apolipoproteins A-I and A-II. 4. The interaction of taurocholate with high-density lipoprotein has been confirmed by density gradient centrifugation using 14C-labelled taurcholate. It is assumed that the interaction of taurocholate with high-density lipoprotein is physiologically of significance.

Adult↗
PubMed1991

Detection of cannabinoid receptors by photoaffinity labelling.

A novel [125I]-labelled photoaffinity ligand designed to detect cannabinoid binding sites has been used in mouse brain preparations and in cultured S49 mouse lymphoma cells. The ligand, 2-iodo-5'-azido-delta 8-THC, shows a high affinity for sites in both brain (Kd = 5.60 pM) and whole cell (Kd = 9.38 pM) systems. Photolabelling studies with brain samples revealed the existence of four ligand-protein adducts, of estimated molecular weights 85.5, 62.1, 30.0 and 25.5 kDa, that were diminished by prior exposure to 8 microM THC. A similar study with S49 cells gave adducts with apparent molecular weights of 62.1, 34.4, 16.9 and 13.5 kDa. The ligand produces a typical cannabinoid cataleptic response in mice suggesting that possibly one or more of the binding sites may be involved in some of the receptor mediated actions of THC.

Affinity Labels↗
PubMed1988

Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A.

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.

Affinity Labels↗
PubMed1987

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label.

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.

3-O-Methylglucose↗
PubMed1990

Detecting precatalytic conformational changes in F1-ATPase with 4-benzoyl(benzoyl)-1-amidofluorescein, a novel fluorescent nucleotide site-specific photoaffinity label.

A novel photoaffinity label for studies with the F1-ATPase has been synthesized and found to be an effective reporter of subunit conformational changes that occur in this enzyme upon multiple nucleotide-binding site occupancy. The new probe, 4-benzoyl(benzoyl)-1-amidofluorescein (BzAF), which possesses structural similarity to purine nucleotides, exhibits bifunctional characteristics that enable it to bind covalently to the exchangeable nucleotide sites on beef heart F1 (via photoactivation of the benzophenone moiety) and, once covalently linked, emit environmentally sensitive fluorescence (via selective excitation of the fluorescein moiety). BzAF binds competitively with ATP in the absence of illumination, with a KI of 50 microM. Under actinic irradiation necessary for generating the covalently reacting diradical triplet state of benzophenone, BzAF behaves as a nucleotide site-directed photoaffinity label of exchangeable (catalytic) sites, and the resulting photoinhibition of ATPase activity displays pseudo first-order rate-saturation kinetics that support formation of a dissociable BzAF.F1 complex (k-1/k1 = 58 microM) prior to covalent binding. The BzAF-induced photoinactivation is protectable with native nucleotide ligand (e.g. MgADP, Kprotect = 0.4 mM). Added corroboration of a catalytic cooperativity mechanism for F1 was obtained by finding a molar stoichiometric ratio [( 3H]BzAF:F1) of 1 required for complete inhibition of ATPase activity. Steady-state fluorescence studies with a unisite-labeled BzAF.F1 complex (a catalytically inactive species on which at least one exchangeable nucleotide-binding site remains unoccupied) display a saturable fluorescence quenching of the bound fluorescein upon titration with MgADP, but no change with MgAMP. These data imply that the filling of more than one of the catalytic binding sites/mol of F1 with nucleotide signals a precatalytic conformational adjustment that is transmitted between catalytic sites and across the beta-alpha-beta domain of the enzyme's subunit structure.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of thyroid hormone receptors.

Photoaffinity label probes of iodothyronines can interact with nuclear receptors in intact cells and in solubilized receptor preparations. These probes have certain advantages over a chemical affinity label in analyzing receptor structure. First, a photoaffinity label probe covalently cross-links only after photoactivation. Therefore, it is possible to demonstrate with appropriate competitive inhibition studies that the photoaffinity label probe associates with the receptor in question. Secondly, since cross-linking only occurs after photolysis, it is possible to adjust the concentration of the photoaffinity label to maximize association with "specific" binding sites relative to "non-specific" associations prior to covalent linkage by photoactivation. The different [125I]iodothyronine-PAL analogues may be useful as probes of the thyroid hormone receptor binding domain since PAL compounds with different affinities for receptor may photocouple to different receptor residues within or proximate to the hormone binding region. These probes may also be useful as an adjunct to receptor purification and in probing the organization of the receptor in chromatin. Lastly, they may provide insights into possible alterations of receptor structure in patients with partial end organ resistance to thyroid hormone (Refetoff et al., 1967; Eil et al., 1982).

Affinity Labels↗
PubMed1991

Azido derivative of tricarboxylic acid for photoaffinity labeling.

A new photoaffinity probe, 5-(1-hydroxy-4-azidophenylazo)-1,2,3-benzenetricarboxylic acid, was synthesized and characterized. This reagent can be potentially used in photoaffinity labeling of the mitochondrial tricarboxylate carrier, as well as of enzymes interacting with tricarboxylic acids. Inhibition and labeling of the mitochondrial tricarboxylate carrier is presented.

Affinity Labels↗
PubMed1988

Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling.

The hydrophobic photoaffinity label 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to label Sendai virus proteins during fusion with cardiolipin and phosphatidylserine liposomes. Preferential labeling of the viral fusion protein during the initial stages of fusion demonstrated that this protein interacts with the hydrophobic core of the target membrane as an initiating event of virus-liposome fusion. Labeling showed time, temperature, and pH dependence consistent with earlier fluorescent measurements of fusion kinetics. The present method provides conclusive evidence supporting the hypothesis that hydrophobic interaction of the fusion protein with the target bilayer is an initial event in the fusion mechanism of viral membranes.

Affinity Labels↗
PubMed1989

Identification of L-glutamate binding sites in chick brain by photoaffinity labeling.

The photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azido benzoate ester, was used to attach L-[3H]glutamate irreversibly to chick brain membranes. Electrophoretic analysis with sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed a major radioactive protein band with an apparent molecular weight of 45,600 +/- 300 Da. A second band with a smaller amount of radioactivity and with an apparent molecular weight of 28,300 +/- 500 Da was also detected. Photolabeling was inhibited by quisqualic acid.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the Ah receptor.

A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected for radiosynthesis with 125I (specific activity 2176 Ci/mmol, equilibrium dissociation constant, KD = 0.76 nM). Following incubation of this 125I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultraviolet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:1 ratio; 3) co-incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a similar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA.

The proteins contacting nascent RNA transcripts in RNA polymerase III transcription complexes have been examined using photoaffinity labeling techniques. The photoaffinity analog 4-S-UTP was incorporated along with [alpha-32P]CTP into VAI transcripts, using a phosphocellulose fractionated HeLa S-100 extract and DNA containing the adenovirus VAI gene. The photoreactive nascent RNA was cross-linked to proximal proteins in the transcription complex. The photoaffinity labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by autoradiography. The specific photoaffinity labeling of RNA polymerase III was dependent on 4-S-UTP and on DNA containing a class III promoter. Photoaffinity labeling was inhibited by 200 micrograms/ml alpha-amanitin. Proteins of 140, 160, 270, and 310 kDa were labeled. These photoaffinity labeled proteins were shown to be stably associated with the DNA template by gel exclusion chromatography. The 160-kDa protein was cross-linked to RNAs approximately 14-18 nucleotides in length, whereas the greater than 250-kDa proteins were cross-linked to RNAs 18-30 nucleotides in length. The 140- and 160-kDa proteins correspond in molecular mass to the two large subunits of RNA polymerase III. The molecular masses of the 270- and 310-kDa proteins, and the length of the RNA cross-linked to them, suggest that these proteins are components of transcription factor (TF) IIIC. These results indicate that the nascent transcript contacts the two largest subunits of RNA polymerase III until the transcription complex reaches the TFIIIC binding site, at which point the nascent transcript contacts TFIIIC.

Adenoviridae↗
PubMed1988

Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label.

A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.

Affinity Labels↗
PubMed1985

Benzodiazepine receptor photoaffinity labeling: correlation of function with binding.

Exhaustive photoaffinity coupling of flunitrazepam to living spinal cord neurons reduced the capacity of benzodiazepines to potentiate the electrophysiologically measured GABA response. In qualitative agreement with reversible binding data the dose-response curve for enhancement of the GABA response by benzodiazepines was shifted to the right, indicating that the remaining reversible benzodiazepine binding sites have lower affinity for benzodiazepines. Photoaffinity labeling did not reduce inhibition of the GABA response by beta-carbolines and there was only a small decrease in beta-carboline binding. In both control and photoaffinity-labeled cultures, the inhibitory effect of beta-carbolines on the GABA response was reversed in the presence of excess benzodiazepine. The results indicate that the effects of photoaffinity labeling are confined to the BZD recognition site, and that coupling between benzodiazepine receptors and GABA receptors remains intact.

Affinity Labels↗
PubMed1992

Sequence analysis of photoaffinity-labelled peptides derived by proteolysis of photosystem-2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine.

Photosystem-2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]-azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron-acceptor site of photosystem 2. SDS/PAGE of the 14C-labelled reaction centres followed by fluorography revealed photoaffinity-labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS-stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8-kDa photoaffinity-labelled peptide, generated by proteolysis of the reaction-centre material with trypsin, was isolated and purified to apparent homogeneity using reverse-phase and size-exclusion HPLC techniques. The amino terminus of the photoaffinity-labelled peptide was determined to be Leu-Gly-Met-Arg-Pro-Xaa-Ile-Ala-Val-Ala-Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu137-Arg225. Chymotrypsin/trypsin digestion of photoaffinity-labelled reaction centres followed by reverse-phase HPLC was used to isolate a smaller photoaffinity-labelled peptide. On Edman sequencing, Ser-Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser-Ala at the amino terminus in the region Leu137-Arg225 are Ser148-Ala-Pro and Ser212-Ala-Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine.

Affinity Labels↗
PubMed1978

Cyclic nucleotide-binding proteins detected by photoaffinity labeling in nucleus and cytoplasm of bovine liver.

A photoaffinity labeling method was used to characterize and compare cyclic nucleotide-binding proteins of bovine liver cytosol with binding proteins of the nucleus. After photoaffinity labeling of cytosol with 8-azido cyclic [(32)P]AMP, autoradiographs of sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed two major labeled proteins of 47,000 and 52,000-55,000 daltons. DEAE-cellulose column-derived fractions suggested that the larger protein was the regulatory subunit of peak II cyclic AMP-dependent protein kinase and the smaller protein was the regulatory subunit of peak I kinase. The smaller protein was largely present as the free regulatory subunit. The two binding proteins differed in their ability to bind cyclic GMP. Binding to both proteins was abolished by excess unlabeled cyclic AMP but not by 5'-AMP. Photoaffinity labeling of a 0.14 M salt extract of nuclei and a nonhistone chromosomal protein preparation revealed two major binding proteins with the same molecular weight and competition profiles as those of the cytosol. Detergent-washed nuclei gave similar results. Several minor binding proteins were observed in both cytosol and nucleus. One protein (36,000 daltons) was unique to the nucleus and had low affinity for 8-azido cyclic AMP. Photoaffinity labeling with cyclic [(3)H]GMP revealed a cytosol protein, absent from the nucleus, of 31,000 daltons and the ligand was competed for by both cyclic GMP and 5'-GMP. These studies suggest that the major specific cyclic AMP-binding proteins of bovine liver are the type I and type II regulatory subunits of cyclic AMP-dependent protein kinase and are present in both nucleus and cytoplasm.

Affinity Labels↗
PubMed1992

CDP-choline: 1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. II. Photoaffinity labeling by radioactive CDP-choline analogs.

Photoaffinity labeling of cholinephosphotransferase from rat liver microsomes directly by its substrate, [32P]CDP-choline or by a synthetic photoreactive CDP-choline analog, 3'(2')-O-(4-benzoyl)benzoyl [32P]CDP-choline (BB-[32P]CDP-choline), was examined for the possible identification of its molecular form on subsequent SDS-PAGE followed by 32P-autoradiography. When the partially purified cholinephosphotransferase was photoirradiated in the presence of [32P]CDP-choline, a considerable amount of 32P-radioactivity was incorporated into the TCA-insoluble component. This incorporation was dependent on irradiation time, Mg2+ or Mn(2+)-requiring and inhibited strongly by the presence of Ca2+. Either CDP-choline or CDP-ethanolamine inhibited the ultraviolet irradiation-dependent incorporation of 32P-radioactivity into the TCA-insoluble component in a dose-dependent manner, whereas neither phosphocholine or 5'-CDP had any effect on this process. These results strongly suggested that the observed 32P-incorporation from [32P]CDP-choline into the protein component could be a consequence of the covalent interaction between cholinephosphotransferase and its substrate, [32P]CDP-choline. Two polypeptides, 25 kDa and 18 kDa, with high 32P-radioactivity were clearly identified on a SDS gel after the direct photoaffinity labeling with [32P]CDP-choline for more than 5 min of ultraviolet irradiation. On the other hand, when BB-[32P]CDP-choline was used as a photoaffinity ligand, a single polypeptide with apparent molecular size of 55 kDa could be rapidly photolabeled within 2.5 min, then this band gradually lost its 32P-radioactivity with increasing time of ultraviolet irradiation. Thus, the overall results strongly indicated that cholinephosphotransferase in rat liver microsomes exists most likely as a 55 kDa polypeptide (or subunit) and that 25 kDa and 18 kDa peptides identified after the direct photoaffinity labeling with [32P]CDP-choline were probably the photo-cleavage products of cholinephosphotransferase during the prolonged ultraviolet irradiation, both of which could contain the catalytic domain of the original enzyme protein(s).

Animals↗