Author index

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as $\text{p-}\text{azido}\text{-m-[}{}^{\mathrm{<mtext>125</mtext><mtext>I</mtext><mtext>]</mtext><mtext>iodobenzylcarazolol</mtext>}}\text{. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su?}$ variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

Biosynthetic relations between protoberberine-, benzo(C)phenanthridine- and B-secoprotoberberine type alkaloids in Corydalis incisa

The biosynthetic relations between protoberberine-, benzo[C]phenanthridine- and B-secoprotoberberine type alkaloids were demonstrated by use of (±)-tetrahydrocoptisine-[8,14-³H HCl, (±)-tetrahydrocorysamine-[8,14-³H]HCl and corynoline-[6-³H]HCl in Corydalis incisa, and the following results were presented. (±)-Tetrahydrocoptisine was converted to corynoline, corydalic acid methyl ester and corydamine hydrochloride. (±)-Tetrahydrocorysamine was converted to corynoline and corydalic acid methyl ester. Evidence that N-methyl-3-[6′-(3′,4′-methylenedioxyphenethylalcohol)]-4-methyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline-[α-³H] HCl was incorporated into corynoline-[11-³H] indicates the occurrence of the ring fission at C₆-N followed by linking ofthe C₆ and C₁₃ positions in (±)-tetrahydrocoptisine and (±)-tetrahydrocorysamine, and suggests the participation of one of two possible intermediates in the biosynthesis of these alkaloids.     

Coupling of the α1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

At rat hepatic membrane α₁-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in α₁-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca²⁺-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (−pi), or in −pi membranes treated with Ca²⁺ (−pi, +Ca²⁺). In these various membrane preparations, different M_r forms of the receptor are also identified by photoaffinity labelling with [¹²⁵I]CP65 526, an aryl azide analog of the α₁-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant M_r = 80 000 subunit is identified in +pi membranes, in −pi membranes a proteolytic M_r = 59 000 fragment is also observed. In −pi, +Ca²⁺ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [¹²⁵I]CP65 526 labelling was determined by laser densitometry scanning and computer analysis. At the M_r = 80 000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in −pi membranes. By contrast, agonist-binding at the M_r = 59 000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved M_r = 59 000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the α₁-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.     

Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

Bakuchalcone,a dihydrofuranochalcone from the seeds of Psoralea corylifolia

A new dihydrofuranochalcone has been identified in seeds of Psoralea corylifolia and its structure confirmed by synthesis.     

Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.     

Isolation and characterization of two forms of Met-tRNAf deacylase from rabbit reticulocyte ribosomes

We have separated and purified two forms of Met-tRNA_f deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes. Deacylase I is a minor activity with an S_20,w of 5.9, D_20,w of 4.9 and M_r of 110 000, while deacylase II is the major activity with an S_20,w of 3.3, D_20,w of 7.1 and M_r of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [³⁵S]Met-tRNA_f to [³⁵S]methionine and tRNA^Met_f and have no effect on reticulocyte [³⁵S]fMet-tRNA_f, [³H]Ala-tRNA or [³H]Lys-tRNA. However, while deacylase I has similar activity throughout the pH range of 6.1–8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1. In addition, deacylase II shows a much greater affinity for pure Met-tRNA_f than deacylase I (K_m of 1.5–3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNA^Met_f, deacylase I is inhibited similarly by any added tRNA.     

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and ¹²⁵I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[¹²⁵I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of ¹²⁵I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of M_r 120 000 was internalized by macrophages somewhat more rapidly than copolymer of M_r 46 000, but was excluded from the yolk sac.     

Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [¹²⁵I]insulin binding to soluble receptors from mouse liver was inhibited by digestion with β-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[³H]hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10^?7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified within two galactoglycoproteins of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with β-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10^?7 M proinsulin slightly hindered labeling while pretreatment with 10^?7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate the glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.     

Photoaffinity labeling of beta-adrenergic receptors: identification of the beta-receptor binding site(s) from turkey, pigeon, and frog erythrocyte

The β-adrenergic receptors in the erythrocyte membranes from turkey, pigeon, and frog have been identified $\text{in situ}$ utilizing the photoaffinity label ±[¹²⁵I]-iodoazidobenzylpindolol, ±[¹²⁵I]IABP. The molecular weights determined by SDS-polyacrylamide gel electrophoresis are the following: turkey, 43,500; pigeon, 53,500, 46,000, and 45,000 [labeled in a ratio of 5 (53,500):2 (46,000 plus 45,000)]; and frog, a broad 60,000 to 67,000 dalton band. The data identify the binding site subunit(s) of these β-adrenergic receptors and suggest that the receptor structure from different β-receptor subtypes and different sources may be different. These biochemical differences may contribute to the pharmacologically observed distinction of β-receptor subtypes.     

Purification and characterization of the messenger ribonucleoprotein-associated casein kinase II of Artemia salina cryptobiotic gastrulae

The mRNP-associated protein kinase is purified to near homogeneity by ion-exchange chromatography on phosphocellulose and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The cyclic nucleotide-independent enzyme phosphorylates casein using either ATP or GTP. The enzyme exists in two forms composed of subunits with M_r 36 500 (α) and 28 000 (β) and of subunits with M_r 36 500 (α), 33 000 (α′) and 28 000 (β). The undegraded enzyme has an M_r of 136 000 ± 7000. The enzyme is inhibited by heparin and hemin and stimulated by spermine. The mRNP-associated protein kinase may be classified as a casein kinase II. Main mRNP protein phosphate acceptors have M_r values of 112 000, 72 000, 65 000, 53 000, 38 000, 28 000, 23 500 and 21 000. Phosphorylation of the M_r 38 000 poly(A)-binding protein resulted in the generation of different acidic ionic species. From the observed inhibition of the translational activity after phosphorylation by the mRNP-associated protein kinase a function in the repression of mRNP is proposed.     

β-adrenegic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0–8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the β-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na⁺K⁺(Mg²⁺)-ATPase activity and a 6–8-fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2–3-fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological β₂ type response with isoproterenol (1.8 · 10^?8 M) > epinephrine (1.1 · 10^?7 M) > norinephrine (3.2 · 10^?6 M). In contrast, binding studies employing (?)-[³H] dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (?)-[³H]dihydroalprenolol by catecholamine agonists.Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of M_r 80 000 and 86 000, whereas in the adult only a single M_r 133 000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.     

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation

Incubating rat fat cell membranes with [³²P]NAD⁺ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42 000, 46 000 and 48 000. Partial proteolytic peptide maps of the M_r = 46 000 and 48 0000 toxin-specific substrates generated by elastase, α-chymotypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the M_r = 42 000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the M_r = 42 000 target displayed a number of peptides which were absent from the maps generated from either the M_r = 46 000 or 48 000 targets. These data suggest that the M_r = 46 000 and 48 000 substrates are closely related proteins, however the relationship between the M_r = 42 000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [³²P]NAD⁺     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.