Translocation of adenosine 3'-phosphate 5'-phosphosulfate into rat liver Golgi vesicles

The translocation of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) across rat liver Golgi-derived vesicles has been studied. Vesicles of the same topographical orientation as in vivo were incubated with a mixture of [adenine-8-3H]PAPS and [35S]PAPS. The tritium to radiolabeled sulfur ratio of the incubation medium was 1.73 +/- 0.03 while that in the vesicles was 1.82 +/- 0.13. This strongly suggests that the entire PAPS molecule was being translocated across the Golgi vesicle membrane even though intact PAPS could not be detected within the vesicles. Translocation of PAPS resulted in accumulation of solutes within vesicles. This accumulation was temperature dependent, saturable (apparent Km = 0.7 microM; Vmax = 25 pmol/mg of protein/10 min), and inhibited by the substrate analogue 3',5'-ADP but not by 2',5'-ADP. Translocation of PAPS was inhibited following treatment of Golgi vesicles with Pronase under conditions in which the activity of a lumenal Golgi membrane marker such as sialyltransferase was not. This result is consistent with the existence of a PAPS carrier protein, portions of which face the cytoplasmic side of the Golgi membrane.     

Characterization of 3''-Phosphoadenosine 5''-Phospho[35S]Sulfate Transport Carrier from Rat Brain Microsomes

3'-Phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS) specific binding properties of rat brain tissue were studied. [35S]PAPS specific binding was optimal at pH 5.8 in either Tris-maleate or potassium phosphate buffers. Association was maximal at low temperature, reaching equilibrium in 20 min. Dissociation was rapid, with a dissociation time of 80 s. Scatchard analysis of [35S]PAPS specific binding was consistent with a single site having a KD of 0.46 +/- 0.06 microM and a Bmax of 20.8 +/- 2.0 pmol/mg of protein. Low concentrations of Triton X-100 (0.025%) were effective in increasing the number of binding sites to a Bmax of 44.5 +/- 4.6 pmol/mg of protein without affecting the affinity. [35S]PAPS specific binding was enriched in crude synaptic membranes (P2) and microsomes (P3). Regional distribution of [35S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [35S]PAPS specific binding in rat brain microsomes was consistent with a membrane protein having a high selectivity for the 3'-O-phosphoryl group substitution on the ribose moiety. Thus, 3'-phosphoadenosine 5'-phosphate was more potent than 2'-phosphoadenosine 5'-phosphate in competing for [35S]PAPS specific binding. Adenosine 5'-phosphosulfate was a good inhibitor of [35S]PAPS specific binding. ATP and ADP were also good displacers. Dipyridamole, a highly selective marker for adenosine uptake sites, was ineffective. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid, the chloride transporter inhibitor, showed an IC50 of 36 +/- 5.1 microM for inhibition of [35S]PAPS specific binding. 2,6-Dichloro-4-nitrophenol had a low selectivity in competing for the [35S]PAPS binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and Localization of the Sulfate-Activating System in Rat Brain

The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta subunit of the insulin receptor is an insulin-activated protein kinase

In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.     

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.     

Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.     

Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.

Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

Adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum: ligand binding properties and the mechanism of substrate inhibition.

[35S]Adenosine-5'-phosphosulfate (APS) binding to Penicillium chrysogenum APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 microM in the presence of 5 mM MgADP. In the presence of 0.1 M (NH4)2SO4, Kd,app was increased to 2.1 +/- 0.7 microM. Bound [35S]APS was displaced by low concentrations of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), or iso-(2') PAPS, or (less efficiently) by adenosine-3,5'-diphosphate (PAP) or adenosine-5'-monosulfate (AMS). The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E. MgADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP). Radioligand displacement was used to verify the Kd for APS dissociation from E.MgADP.APS and to determine the Kd values for the dissociation of iso-PAPS (13 +/- 5 microM), PAP (4.8 mM), or AMS (5.2 mM) from their respective ternary enzyme.MgADP.ligand complexes. Incubation of the fungal enzyme with [gamma-32P]MgATP did not yield a phosphoenzyme that survives gel filtration or gel electrophoresis.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate

Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited. Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP. The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer. Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating. Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide. Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS. In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies. The primary kinetic mechanism in the forward direction is sequential. Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS. At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism. The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS. The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.     

Light stimulates phosphorylation of two large membrane proteins in frog photoreceptors

By photoactivating rhodopsin, light indirectly initiates a series of biochemical reactions within photoreceptors as part of the visual process. I herein report that one of the light-stimulated reactions in bullfrog photoreceptors is the phosphorylation of two previously unreported proteins (220 and 240 kDa). Their phosphorylation by endogenous kinase(s) is readily observed in freshly isolated, fragmented rods. On subcellular fractionation, the labeled proteins copurify with the membranes of the outer segments, from which they cannot be extracted with low ionic strength. They appear to be integral membrane proteins of the disk or plasma membranes. Their light-induced phosphorylation is also observed in intact receptors when excised frog retinas are incubated under in vivo conditions with 32PO4. Thus, appropriate kinase(s) is (are) present within outer segments and presumably is (are) the one(s) responsible for phosphorylation in fragmented cells. In the presence of adenosine 5'-(gamma-[35S]thiotriphosphate) [( 35S] ATP-gamma-S), light can also stimulate thiophosphorylation, leading to preferential labeling of the 220-kDa protein. On the basis of four criteria (electroporetic mobility, membrane location, binding of concanavalin A, and mobility shifts with SH oxidation), the 220-kDa protein appears to correspond to the membrane protein previously identified at the rims of rod disks [Papermaster, D.S., Schneider, B.G., Zorn, M.A. & Kraehenbuhl, J.P. (1978) J. Cell Biol. 78, 415-425]. Identity of the other substrate protein is unknown. When fragmented cells are illuminated with a flash of 1-ms duration, the half-time for phosphorylation is about 1 min with ATP at 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5'-Phosphosulfate Kinase

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp(136), which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.     

The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. M?ssbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.     

Light-regulated biochemical events in invertebrate photoreceptors. 1. Light-activated guanosinetriphosphatase, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes

The occurrence of a guanine nucleotide binding protein activated by squid rhodopsin was established by examination of GTPase activity, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes. Purified squid (Loligo opalescens) photoreceptors exhibited GTPase activity that increased 3-4-fold by illumination. Half-maximal GTPase activity was observed when 2% of the rhodopsin was photoconverted to metarhodopsin. The Km of the light-regulated activity was 1 microM GTP. Binding of the hydrolysis-resistant GTP analogue guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] was enhanced greater than 10 times by illumination. A protein, Mr 44 000, was identified as a component of the light-activated guanine nucleotide binding protein/GTPase through its specific labeling with [32P]NAD catalyzed by cholera toxin: light increased the extent of 32P incorporation 7-fold. The addition of ATP to the membrane suspension enhanced labeling, while guanine nucleotides inhibited labeling with the relative potency GTP gamma S much greater than GDP greater than GTP greater than Gpp(NH)p. The 44 000-dalton protein was membrane bound irrespective of variations in ionic strength and divalent ion concentration over a wide range. These results suggest that a G protein, which incorporates both GTP binding and hydrolysis functions, is intimately involved in the visual process of invertebrate photoreceptors.     

Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.     

ATP binding/hydrolysis by and phosphorylation of peroxisomal ATP-binding cassette proteins PMP70 (ABCD3) and adrenoleukodystrophy protein (ABCD1)

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP when peroxisomes were incubated with these nucleotides at 37 degrees C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[gamma-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[alpha-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.     

Sulfohydrolytic degradation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and adenosine 5'-phosphosulfate (APS) by enzymes of a nucleotide pyrophosphatase nature

The sulfohydrolytic activity to degrade active sulfate (3'-phosphoadenosine 5'-phosphosulfate, PAPS) and its precursor, APS (adenosine 5'-phosphosulfate), with a pH optimum at 9.5 was found to be widely distributed in various tissues of rats. In the liver, the activity was located in plasma membranes and endoplasmic reticula. Triton X-100 solubilized rough and smooth endoplasmic reticula gave two peaks of the activity on gel filtration, both of which had nucleotide pyrophosphatase activities, hydrolyzing the pyrophosphate linkages of ATP, NAD, and UDP-Glc, and the phosphodiester linkage of PNTP (p-nitrophenyl-thymidine 5'-monophosphate) besides PAPS and APS.     

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding

Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.