Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.     

Analysis of protein incorporation of radioactive isotopes in the Chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis

The patterns of [3H]-leucine and [32P]-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of [3H]-leucine- and [32P]-phosphate-labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.     

The preparation and use of pyridoxal [32P]phosphate as a labeling reagent for proteins on the outer surface of membranes.

Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Phorbol ester, calcium ionophore, or serum added to quiescent rat embryo fibroblast cells all result in the elevated phosphorylation of two 28,000-dalton mammalian stress proteins

Rat embryo fibroblast cells grown under stress (e.g. heat shock, arsenite, or amino acid analogue treatment) show elevated levels of a number of proteins with apparent molecular masses between 28,000-110,000 daltons (i.e. stress proteins). It is shown that the smaller 28,000-dalton stress proteins, which do not contain methionine, are comprised of at least four isoforms, all of which appear related as determined by one-dimensional peptide mapping. [32P]H3PO4 labeling of normal and stressed cells demonstrates that three of the four 28-kDa isoforms are phosphoproteins. In the course of other studies phosphorylation of two 28,000-dalton proteins was observed in quiescent rat embryo fibroblasts following the addition of either the phorbol diester, phorbol-12-myristate-13 acetate, a calcium ionophore, A23187, or simply fresh serum. It is shown here that these two 28,000-dalton proteins are in fact two of the 28 kDa mammalian stress proteins.     

Guanylylation and adenylylation of the alpha regulatory proteins of herpes simplex virus require a viral beta or gamma function.

Herpes simplex virus genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Most of the products of the first group, the alpha genes, appear to have regulatory functions. We report that the alpha proteins, infected cell proteins 4, 0, 22, and 27 of herpes simplex virus 1 and 4, 0, and 27 of herpes simplex virus 2, were labeled in the isolated nuclei of infected HeLa cells with [alpha-32P]GTP or [alpha-32P]ATP late in infection and that these proteins represent the largest group of virus-specific proteins labeled in this fashion. Studies with [2-3H]ATP, in which the label is in the purine ring, showed that a portion of the label in alpha proteins and in at least one other infected cell protein is due to nucleotidylylation. Analyses of the labeling reactions in nuclei of (i) cells infected with temperature-sensitive mutants at nonpermissive temperatures, (ii) cells infected with wild-type virus and harvested at different times postinfection, and (iii) cells treated with inhibitors of protein synthesis or of synthesis of viral DNA led to the conclusion that viral gene functions expressed after the synthesis of alpha proteins are required for the labeling of the alpha proteins with [alpha-32P]GTP. We conclude that several of the alpha proteins are extensively posttranslationally modified and that these modifications include nucleotidylylation.     

Small GTP‐binding proteins in the nuclei of human placenta

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [α³²P]‐GTP. Prior to this study the number of GTP‐binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP‐binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [α³²P]‐GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [α³²P]‐GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP‐binding proteins. The GTP‐binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP‐binding proteins in the development and physiology of the placenta. J. Cell. Biochem. 84: 100–107, 2002. © 2001 Wiley‐Liss, Inc.     

Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].     

Use of 8-azidoguanosine 5'-[gamma-32P]triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

In an in vitro incubation, 8-azidoguanosine 5'-[gamma-32P]triphosphate ( [gamma-32P]-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with [gamma-32P]-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, [gamma-32P]-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by [gamma-32P]-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.     

Induction of 32- and 34-kDa stress proteins by sodium arsenite, heavy metals, and thiol-reactive agents

Challenge of human or murine melanoma cells with sodium arsenite, heavy metals (Zn2+, Cu2+ and Cd2+), or thiol-reactive agents (p-chloromercuribenzoate and iodoacetamide) induced the synthesis of four stress proteins with molecular masses of 100, 90, 72 (a doublet), and 32 (human) or 34 (murine) kDa. Enhanced expression of the 32- and 34-kDa polypeptides (p32 and p34) preceded or paralleled the synthesis of the other stress proteins. Hyperthermia, the calcium ionophore A23187, and amino acid analogs (L-azetidine-2-carboxylic acid and L-canavanine) induced the formation of the major stress proteins, but failed to increase synthesis of p32 and p34. Characterization of the dose and time dependence of p32 and p34 synthesis in human (A375) and murine (B16-F10) melanoma cells, respectively, indicated that these proteins were subject to similar regulatory mechanisms. Electrophoretic analysis of stressed cells pulsed with different metabolic precursors revealed that p32 and p34 were radiolabeled with [35S]methionine or 3H-amino acids but not by [3H]mannose or [35S]cysteine. Polyclonal antibodies raised against human p32 cross-reacted with murine p34. These data suggest that p32 and p34 are closely regulated human and murine gene products, respectively, whose synthesis can be modulated by thiol-reactive reagents. Induction of p32 and p34 by these agents, but not by heat shock, suggests that these proteins are a subset of stress-inducible gene products.     

Biosynthesis of prostatic acid phosphatase in a normal human cell-line

The biosynthesis of the prostatic form of human acid phosphatase was studied in normal embryonic lung cells, WI-38, by metabolic labeling with tritiated leucine and [32P]phosphate, followed by specific immunoprecipitation, gel electrophoresis, and fluorography. Of the total tartrate-inhibitable acid phosphatase activity in WI-38 cells, 30% is due to the prostatic form. The primary translation product that leads eventually to the mature prostatic enzyme is a precursor polypeptide of 112 kDa. The precursor polypeptide is processed to mature polypeptides of 59, 55, and 49 kDa via an intermediate 91-kDa precursor. WI-38 cells also secrete a 113-kDa peptide into the medium. The precursor and mature polypeptides are glycosylated and phosphorylated. Upon treatment with endo-beta-hexosaminidase H, the apparent molecular weighs of the polypeptides are reduced by approximately 4 kDa and phosphate is lost.     

Isolation and immunological characterization of the mammalian 32-/34-kDa stress protein

Challenge of mammalian cells with heavy metals or sulfhydryl-reactive agents including sodium arsenite induces the de novo synthesis of a 32-/34-kDa stress protein (p32) (M. M. Caltabiano, T. P. Koestler, G. Poste, and R. G. Greig (1986) J. Biol. Chem. 261, 13,381). Here we report that antibody prepared against p32/p34 purified from human A375 melanoma cells immunoprecipitated an antigen of similar molecular mass from a panel of human, rat, and murine cells following challenge with sodium arsenite. No reactivity was observed in lysates from control, uninsulted cultures. The precise molecular mass of the arsenite-induced antigen was species-specific: 32 kDa (human and rat) and 34 kDa (murine). Indirect immunofluorescence analysis using affinity-purified monospecific IgG demonstrated that p32/p34 was localized to the cytoplasm and displayed a perinuclear distribution.     

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.     

Phosphate transport and proteins with SH groups in rat liver mitochondria

Phosphate transport in rat liver mitochondria was studied by following [32P] phosphate uptake within physiological concentrations. Transport inhibition due to mersalyl and protection by mersalyl against N-ethylmaleimide measured in those conditions corresponded to earlier results obtained by the swelling technique. When mitochondria were incubated with [3H] N-ethylmaleimide in the presence of mersalyl, the radioactive labeling in proteins of particles obtained after sonication was decreased in all fractions, but three proteins were both highly alkylated and also highly protected by mersalyl (M.W. 48,000 - 36,000 - 31,000). Two of these (M.W. 36,000 and 31,000) were partially purified by ultrogel chromatography in the presence of sodium dodecyl sulfate. Furthermore, it was shown that both phosphate and nigericin diminished labeling by N-ethylmaleimide in the final supernatant fraction. Two proteins (M.W. 98,000 and 31,000) were significantly alkylated by [3H] N-ethylmaleimide and protected by phosphate and nigericin.     

Phosphoproteins crosslinked to poly(A) + heterogeneous nuclear RNA after irradiation with ultraviolet light

Isolated liver nuclei or whole lymph node lymphocytes stimulated with concanavalin A in culture were irradiated with ultraviolet light. The crosslinked structures of poly(A)+ heterogeneous nuclear RNA and protein were purified on oligo(dT)-cellulose after labelling irradiated nuclei in the presence of adenosine 5'-[gamma-32P]triphosphate and analysed by SDS-polyacrylamide gel electrophoresis. The liver and lymphocyte nuclear proteins included about 17-19 species of 35-150 kDa and were shown to produce quite similar electrophoretic band patterns. Two proteins of 110-120 and 40-42 kDa were phosphorylated. Using partial proteolytic digestion the large-size crosslinked phosphoprotein has been identified as the 110 kDa component described previously (Schweiger, A. and Kostka, G. (1984) Biochim. Biophys. Acta 782, 262-268). The 40-42 kDa band was presumably related to the group C species of main proteins associated with heterogeneous nuclear RNA. In crosslinked nuclear structures from rats treated with low doses of alpha-amanitin for 1 h the relative amount of the 110-120 kDa phosphoprotein was reduced while the labelling with [32P]ATP was almost abolished.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

The effect of isoproterenol and hydroxyurea on the presence of ubiquitin and protein A24 in the rat salivary gland

The in vivo administration of hydroxyurea for 12 h counteracts DNA synthesis and cell cycling stimulated by 72 h of isoproterenol treatment in rat salivary gland, as determined by fluorescence-activated flow cytometry. Hydroxyurea has little effect on [³H]leucine incorporation (protein synthesis) of the nuclear proteins soluble in 0.35 M NaCl, when examined by polyacrylamide gel chromatography and autoradiography from electro-statically sorted nuclei of (G₀+G₁) and (G₂+M) phases of the in vivo cell cycle. Differential incorporation of [³H]leucine into nuclear proteins was observed during various phases of the cell cycle. Proteins ‘X’ and ‘Z’, observed in stained gel chromatographs of the 0.35 M NaCl-soluble nuclear proteins, were identified by biochemical analyses as ubiquitin and protein A₂₄, respectively. Ubiquitin appeared transiently while A₂₄ increased in gel chromatograms concomitant with progressive quiescence of the salivary gland induced by hydroxyurea.     

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.     

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).