Studies of Sulfate Utilization by Algae. 6. Adenosine-3'-Phosphate-5'-Phosphosulfate (PAPS) as an Intermediate in Thiosulfate Formation From Sulfate by Cell-Free Extracts of Chlorella

When cell-free preparations of Chlorella pyrenoidosa Chick (Emerson strain 3) form thiosulfate from labeled sulfate, another radioactive compound also appears. This compound has been isolated in quantity and is shown to be identical with adenosine-3′-phosphate-5′-phosphosulfate (PAPS) on the basis of its chromatographic and electrophoretic behavior, chemical composition, sensitivity to selective degradative enzymes, and its ability to serve as a substrate for rat liver aryl sulphotransferase. In addition, as expected for PAPS, the compound on mild acid treatment yields all of its radioactive sulfur as sulfate, and is converted to a compound identical with adenosine-3′,5′-diphosphate (PAP). Replacement of sulfate and ATP by this PAP³⁵S in the usual incubation mixture yields the same product, thiosulfate, which can be isolated as such or detected as acid-volatile radioactivity. This conversion of PAP³⁵S to thiosulfate still requires the addition of Mg²⁺ and a reductant such as 2,3-dimercaptopropan-1-ol (BAL). The cause of our previous result that high concentrations of ATP inhibit thiosulfate formation from sulfate can be ascribed to a small amount of PAP contaminating the ATP preparations, since PAP proves to be an exceedingly effective inhibitor of the conversion of PAP³⁵S to thiosulfate. Sulfate reduction to thiosulfate by Chlorella extracts is discussed and compared with similar systems from other organisms.     

Phenolsulphotransferase activity in the developing mosquito (Aedes togoi)

Phenolsulphotransferase (PST) activity was measured with N-acetyldopamine (NADA) and harmol as substrates in the larvae, pupae and adult mosquito (Aedes togoi). Only the newly emerged pupae showed high PST activity 1–4 hr after pupation. PST activity could also be measured in each individual pupa, with the female exhibiting significantly higher specific activity (30 ± 3.7 pmol NADA [³⁵S]sulphate/min per mg protein) than the males (13.6 ± 2.9 pmol NADA [³⁵S]sulphate/min per mg protein). The optimum pH for the PST reaction was 9.0. The K_m values for [³⁵S]PAP were 0.55 and 2.5 μM when measured with NADA and harmol as acceptors, respectively; the corresponding K_m values for these two substrates were 2.61 and 16.1 μM. Studies with 2,6-dichloro-4-nitrophenol showed a dose-dependent inhibition of PST. Sulphate conjugation of NADA from ATP and sodium [³⁵S]sulphate was also demonstrated with pupal extracts, with pH optimum between 8.6 and 9.0. The specific activity of this overall sulphate conjugation, measured in the female pupal extract was 5.08 pmol NADA [³⁵S]sulphate/min per mg protein and 1.68 pmol harmol [³⁵S]sulphate/min per mg protein. The importance and possible function of sulphate conjugation of NADA in insects is discussed.     

Enzymatic sulfation of a large molecular weight glycoprotein associated with pig brain membranes

Pig brain membranes catalyze the transfer of [³⁵S]sulfate from 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate into two macromolecular endogenous acceptors. Several operational enzymatic properties of the sulfotransferase activity have been defined. An apparent K_m = 0.65 μm for 3′-phosphoadenosine 5′-phosphosulfate has been determined for the pig brain in vitro sulfotransferase system. Direct proof for the absolute requirement of the 3′-phosphate moiety of 3′-phosphoadenosine 5′-phosphosulfate is presented. The nucleotide end product, 3′,5′-ADP, is a potent competitive inhibitor of the pig brain sulfotransferase activity. One of the major products enzymatically labeled during incubation with 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate is a membrane-bound glycoprotein of high molecular weight. The sulfated glycoprotein appears to be an integral membrane glycoprotein, requiring 1% Triton X-100 for extraction. An ³⁵S-labeled oligosaccharide, released by mild base treatment, contains O-sulfate ester groups and at least one N-acetylneuraminic acid residue. The sulfated glycoprotein has an apparent molecular weight of 198,000. Under the same in vitro conditions [³⁵S]sulfate is also incorporated into a membrane-associated ³⁵S-labeled proteoglycan having the properties of heparan sulfate. The ³⁵S-labeled proteoglycan is electrostatically bound to the pig brain membranes, and can be readily extracted with 1 m NaCl.     

Rapid and simple measurement of ATP-sulfurylase activity in crude plant extracts using an ATP meter for bioluminescence determination

ATP-sulfurylase (EC 2.7.7.4.) catalyzes the first step in assimilatory sulfate reduction, forming adenosine 5′-phosphosulfate (APS) and pyrophosphate from ATP and SO₄^2?. The extractable activity of ATP-sulfurylase was determined in crude extracts from Phaseolus vulgaris by measuring the formation of ATP, produced in the reverse reaction from APS and pyrophosphate, using purified luciferase and luciferin in an ATP meter. One determination can be performed per minute. The rates of ATP-sulfurylase activity determined by this method were about 25 times higher than the ones measured in the forward reaction as AP³⁵S formed from ATP and ³⁵SO₄^2?.     

Sulfhydrolase Activity in Sediments of Wintergreen Lake, Kalamazoo County, Michigan

The hydrolysis of p-nitrophenyl sulfate, p-nitrocatechol sulfate, and [³⁵S]sodium dodecyl sulfate was examined in anoxic sediments of Wintergreen Lake, Michigan. Significant levels of sulfhydrolase activity were observed in littoral, transition, and profundal sediment samples. Rates of sulfate formation suggest that the sulfhydrolase system would represent a major source of sulfate within these sediments. Sulfate formed by ester sulfate hydrolysis can support dissimilatory sulfate reduction as shown by the incorporation of ³⁵S from labeled sodium dodecyl sulfate into H₂³⁵S. Sulfhydrolase activity varied with sediment depth, was greatest in the littoral zone, and was sensitive to the presence of oxygen. Estimations of ester sulfate concentrations in sediments revealed large quantities of ester sulfate (~30% of total sulfur). Both total sulfur and ester sulfate concentrations varied with the sediment type and were two to three orders of magnitude greater than the inorganic sulfur concentration.     

The effect of chronic ethanol consumption on temperature-dependent physical properties of liver mitochondrial membranes

We have reported that the monovalent ionophore monensin causes undersulfated chondroitin sulfate biosynthesis in cultured chondrocytes. In order to clarify the mechanism of this diminished sulfation, we have measured the rate of incorporation of sulfate into chondrocytes and assayed the cellular ATP levels. We have also measured sulfatase activity, the incorporation of ³⁵SO₄ into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and endogenous sulfotransferase activity in the cell-free extracts. We find that: (1) The incorporation of ³⁵SO₄ into the free sulfate pool in chondrocytes was not inhibited by monensin. (2) The ATP levels of monensin-treated chondrocytes were the same as control cells. (3) There was no sulfatase activity in both control and monensin-treated chondrocytes. (4) Enzymatic analyses revealed that ³⁵SO₄ incorporation into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and subsequent sulfotransferase activity were not inhibited in the presence of monensin. At present the most tenable hypothesis to account for monensin causing undersulfated chondroitin sulfate synthesis is that the ionophore impairs the access of proteoglycans to the sulfotransferases in the luminal walls of the Golgi structures.     

Studies of sulfate utilization by algae: 10. Nutritional and enzymatic characterization of chlorella mutants impaired for sulfate utilization

Seven mutants of Chlorella pyrenoidosa (Emerson strain 3) impaired for sulfate utilization have been isolated after treatment of the wild-type organism with nitrosoguanidine by replica plating on media containing thiosulfate and l-methionine. These mutants fall into three classes based on their ability to grow on sulfate, accumulate compounds labeled from sulfate-³⁵S, and reduce adenosine 3′-phosphate 5′-phosphosulfate-³⁵S (PAPS-³⁵S) to thiosulfate-³⁵S. Mutant Sat₂⁻ cannot grow on sulfate, but it accumulates thiosulfate-³⁵S and homocysteic acid-³⁵S from sulfate-³⁵S in vivo. In addition, extracts of mutant Sat₂⁻ reduce PAPS-³⁵S to thiosulfate-³⁵S, indicating the possession of enzyme fractions S and A, both of which are required for thiosulfate formation. Mutants Sat₁⁻, Sat₃⁻, Sat₄⁻, Sat₅⁻, and Sat₆⁻ cannot grow on sulfate, and their extracts lack the ability to reduce PAPS-³⁵S to thiosulfate-³⁵S. Mutant Sat₇⁻R₁, a probable revertant, can grow on sulfate but still lacks the ability to reduce PAPS-³⁵S to thiosulfate-³⁵S in vitro. Complementation experiments in vitro show that the block in formation of acid-volatile radioactivity in every case is due to the absence of activity associated with fraction S. All mutants can grow on thiosulfate and all possess the activating enzymes which convert sulfate to PAPS. Through a comparison of nutritional and enzymatic characteristics, the first outlines of a branched and complicated pathway for sulfate reduction in Chlorella are beginning to emerge.     

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic,intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP³⁵S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (K_m 5 to 50 μM) and a low affinity reaction (K_m 360 to 2,900 μM). The efficiency of sulfation (V_max/K_m) for both reactions was 5-fold higher for (+)- than for (?)-ISO. When the hepatic PSTs were resolved by ionexchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a V_max/K_m value 6.1-fold higher for (+)- than for (?)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a V_max/K_m value 5.7-fold higher for (+)- than for (?)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (?)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation. © 1993 Wiley-Liss, Inc.     

Suppression of cardiac phosphatidate phosphohydrolase 1 activity and lipin mRNA expression in Zucker diabetic fatty rats and humans with type 2 diabetes mellitus

Lipin functions in mammalian phospholipid biosynthesis through its phosphatidate phosphohydrolase 1 (PAP₁) activity. Here, we studied cardiac PAP₁ activity and lipin expression ex vivo in 8-month-old Zucker diabetic fatty (ZDF) rats and humans with type 2 diabetes mellitus undergoing open heart surgery for coronary bypass grafting. Compared to non-diabetic littermates (ZDF-fa/+), left ventricular PAP₁ activity was 29% lower in diabetic ZDF-fa/fa rats. Left ventricular PAP₁ activities were 2.1-fold (ZDF-fa/fa) and 3.6-fold (ZDF-fa/+) higher than the respective atrial activities, indicating marked differences in cardiac distribution of PAP₁. PAP₁ activity was highly related with cardiac lipin-1 and lipin-3 mRNA expression in ZDF rats (r = 0.99 and 0.96). Consistent with the findings in experimental animals, human atrial tissue displayed PAP₁ activity that was 33% lower in those having diabetes than in non-diabetic controls. Accordingly, atrial lipin-1 and lipin-3 mRNA expression in diabetic patients was 50% and 59% lower as in non-diabetic patients, respectively. Insulin therapy increased both PAP₁ activity and lipin mRNA expression in diabetic patients. We conclude that suppression of cardiac PAP₁ activity/lipin expression may contribute to metabolic dysfunction of the diabetic heart.     

Characterization of ecdysone 20-monooxygenase activity in wandering stage larvae of Drosophilamelanogaster: Evidence for mitochondrial and microsomal cytochrome P-450 dependent systems

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone to 20-hydroxyecdysone, was characterized in wandering stage larvae of Drosophila melanogaster using an in vitro radioassay in conjunction with analytical thin layer chromatography. 20-Hydroxyecdysone was confirmed to be the product of the enzyme radioassay system by high pressure liquid chromatography. The 20-monooxygenase was found to be most active in a 0.10 M phosphate buffer, pH 7.5, was inhibited by Ca²⁺, Mg²⁺ and Se⁴⁺ and exhibited a temperature optimum at 35°C. Differential centrifugation, sucrose step gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with both the mitochondrial and microsomal fractions. Substrate kinetics experiments indicated that the mitochondrial and microsomal monooxygenase systems exhibit apparent K_ms for ecdysone of 6.4 × 10⁻⁸ and 9.9 × 10⁻⁸ M, respectively, with apparent V_maxs of 4.1 and 10.2 pg 20-hydroxyecdysone formed/min per mg tissue equiv., respectively. Both monooxygenase systems were inhibited by their product 20-hydroxyecdysone. The cytochrome P-450 nature of these insect steroid hydoxylases was initially suggested by their requirement for NADPH, NADH was approximately half as effective in supporting the mitochrondrial monooxygenase activity. In addition, both monooxygenase systems were inhibited by carbon monoxide, ellipticine, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Photochemical action spectra of ecdysone 20-monooxygenase activity confirmed the cytochrome P-450 dependency of both the mitochondrial and microsomal ecdysone 20-hydroxylase systems.     

Design and synthesis of structurally defined heparan sulfate (HS)-FK506 conjugates as an exogenous approach to investigate biological functions of nucleus HS

Although heparan sulfate (HS) is widely implicated in numerous physiological and pathological processes, the biological function of nucleus HS remains underexplored, largely due to its complex structure and high hydrophilic property. To supplement these efforts, ideal vehicles are drawing attention as they combine attractive features including lipid solubility for penetrating cell membrane, high affinity binding to its target receptor, metabolic stability, and no cellular actions resulting in toxicity. Herein, we develop a convenient and promising strategy to prepare HS-FK506 conjugates for membrane transport and entry into nucleus, where click chemistry takes easily place between the exocyclic allyl group of a clinic drug FK506 and thiol as a handle incorporated into HS analogues. HS derivatives for constructing the conjugates were synthesized using a cutting-edge chemoenzymatic method. Meantime, [³⁵S] labeled 3′-phosphoadenosine 5′-phosphosulfate (PAP³⁵S) and [¹⁴C] glucuronic acid (Glc A) were adopted to label HS-FK506 conjugates, respectively, to evaluate their efficiency of nucleus entry, as a result, ¹⁴C Glc A was sensitive, effective and reliable whereas PAP³⁵S gave rise to a mixture of labeled compounds, hampering the understanding of structure-function relationship of nucleus HS. Compared with the corresponding HS, the amount of HS-FK506 conjugates to translocate into nucleus from radioactive assay experiments sharply increased, e.g. tridecasaccharide-FK506 1d increased by approximate 10 folds, offering a simple and robust platform for enabling hydrophilic compounds including carbohydrates to translocate into nucleus and shedding light on their biological functions.     

Ecdysone metabolism in Pieris brassicae during the feeding last larval instar

Ecdysone metabolism in Pieris brassicae during the feeding last larval stage was investigated by using ³H-labeled ecdysteroid injections followed by high-performance liquid chromatographic (HPLC

1 Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; E = ecdysone; Eoic = ecdysonoic acid; 2026E′ = 3-epi-20,26-dihydroxyecdysone; E′ = 3-epiecdysone; E′oic = 3-epiecdysonoic acid; E′8P = 3-epiecdysone 3-phosphate; 20E′ = 3-epi-20-hydroxyecdysone; 20E′3P = 3-epi-20-hydroxyecdysone 3-phosphate; FT = Fourier transform; HPLC = high-performance liquid chromatography; 20E = 20-hydroxyecdysone; 20Eoic = 20-hydroxyecdysonoic acid; NMR = nuclear magnetic resonance; NP-HPLC = normal phase HPLC; RP-HPLC = reverse phase HPLC; TFA = trifluoroacetic acid; Tris = tris(hydroxymethyl)-aminomethane.

) analysis of metabolites. Metabolites were generally identified by comigration with available references in different HPLC systems. Analysis of compounds for which no reference was available required a large-scale preparation and purification for their identification by ¹H nuclear magnetic resonance spectrometry. The metabolic reactions affect the ecdysone molecule at C-3, C-20, and C-26, leading to molecules which are modified at one, two, or three of these positions. At C-20, hydroxylation leads to 20-hydroxyecdysteroids. At C-26, hydroxylation leads to 26-hydroxyecdysteroids which can be further converted into 26-oic derivatives (ecdysonoic acids) by oxidation. At C-3, there are several possibilities: there may be oxidation into 3-dehydroecdysteroids, or epimerization possibly followed by phosphate conjugation. Thus, injected 20-hydroxyecdysone was converted principally into 20-hydroxyecdysonoic acid, 3-dehydro-20-hydroxyecdysone, and 3-epi-20-hydroxyecdysone 3-phosphate. Labelled ecdysone mainly gave the same metabolites doubled by a homologous series lacking the 20-hydroxyl group.     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly,Sarcophaga peregrina

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC₅₀=0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3′-phosphoadenosine 5′-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.     

Conversion of ecdysone and 20-hydroxyecdysone into 3-dehydroecdysteroids is a major pathway in third instar Drosophila melanogaster larvae

Ecdysone and 20-hydroxyecdysone metabolism was investigated in third instar Drosophila larvae both in vivo by injecting radiolabelled ecdysteroids and in vitro by incubating various tissues with labelled ecdysteroids.Ecdysone metabolism proceeds through different pathways: (1) C-20 hydroxylation; (2) C-26 hydroxylation and C-26 oxidation leading to the formation of 26-hydroxyecdysteroids (26-hydroxyecdysone and 20,26-dihydroxyecdysone) and acidic compounds (ecdysonoic acid and 20-hydroxyecdysonoic acid); C-3 oxidation and C-3 epimerization then conjugation leading to the formation of 3-dehydrocompounds (3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone), 3-epimers (3-epiecdysone and 3-epi-20-hydroxyecdysone) and conjugates (only one conjugate was tentatively characterized as 3-epi-20-hydroxyecdysone-3-phosphate). 3-Dehydrocompounds are the major metabolites formed in third instar Drosophila larvae and C-3 oxidation occurs in various tissues. Experiments using tritiated cholesterol provided evidence that 3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone are true endogenous ecdysteroids in Drosophila larvae.     

Characterization of acetyl-CoA: Ecdysone 3-acetyltransferase in Schistocerca gregaria larvae

Characterization of the acetyltransferase (acetyl-CoA: ecdysone 3-acetyltransferase) which catalyzes the conversion of ecdysone into ecdysone 3-acetate was carried out in gastric caecae of day 7 last instar larvae of Schistocerca gregaria. This enzyme is one of the enzymic systems involved in the inactivation of ecdysteroids. The acetyltransferase exhibited a microsomal subcellular localization, an apparent K_m for ecdysone of 71 μM, a maximal specific activity of 7.2 nmol/min/mg of protein and was inhibited competitively in the presence of 20-hydroxyecdysone with K_i = 68.8 μM. The enzyme required acetyl-CoA as co-substrate for its activity, the apparent K_m for acetyl-CoA being 47.2 μM. Acetic acid could not replace acetyl-CoA as the co-substrate, indicating that the enzyme is an acetyl-CoA: ecdysone acetyltransferase and not a hydrolase. Similarly, esterification of ecdysone was not observed when long-chain fatty acyl-CoA derivatives were substituted as co-substrates. The reaction was linear for 20 min and with protein concentration up to 0.8 mg/ml.The formation of 20-hydroxyecdysone 3-acetate has been demonstrated in the same microsomal fraction and required also acetyl-CoA as co-substrate. The apparent K_m of the acetyltransferase for 20-hydroxyecdysone was 53.5 μM, revealing that the enzyme had a somewhat stronger affinity for 20-hydroxyecdysone than for ecdysone.     

The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes

The synthesis and degradation of 5′-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24–36 h in culture. After a 30 h lag period 5′-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5′-nucleotidase in the cultured hepatocytes using [³⁵S]methionine showed that the 5′-nucleotidase monomer was synthesised as an M_r 67 000 form which was converted to the mature M_r 72 000 form. [³⁵S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an M_r 57 000 form. The immature M_r 67 000 form of 5′-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5′-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.     

Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin

Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [³⁵S]cysteine, in which [³⁵S]sulfide and iron are incorporated into Fd to build up the ³⁵S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [³⁵S]sulfide or iron, or in Fe-S cluster formation itself. [³⁵S]Sulfide was liberated from [³⁵S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [³⁵S]cysteine occurred before the formation of the ³⁵S-labeled Fe-S cluster, and the amount of radioactivity in [³⁵S]sulfide was greater than that in ³⁵S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na₂S) inhibited competitively formation of the ³⁵S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [³⁵S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.     

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the α and β subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [³⁵S]sulfate was also incorporated into several other proteins in addition to LH. The location of ³⁵SO₄^2? in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.     

Oxidation and reduction of sulfite by chloroplasts and formation of sulfite addition compounds

After exposing intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) and capable of photoreducing CO₂ at high rates to different concentrations of radioactive sulfite in the light or in the dark, ³⁵SO₂ and H₂³⁵S were removed from the acidified suspensions in a stream of nitrogen. Remaining activity could be fractionated into sulfate, organic sulfides, and sulfite addition compounds. When chloroplast suspensions contained catalase, superoxide dismutase and O-acetylserine, the oxidation of sulfite to sulfate was slower in the light than the reductive formation of sulfides that exhibited a maximum rate of about 2 micromoles per milligram chlorophyll per hour, equivalent to about 1% of maximum carbon assimilation. Botht the oxidative and the reductive detoxification of sulfite were very slow in the dark. Oxidation was somewhat, but not much, accelerated in the light in the absence of O-acetylserine, which caused a dramatic decrease in the formation of organic sulfides and an equally dramatic increase in the concentration of sulfite addition compounds whose formation was light-dependent. The sulfite addition compounds were not identified. Addition compounds did not accumulate in the dark. In the light, the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, decreased not only the reduction, but also the oxidation of sulfite and the formation of addition compounds.