The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells

Balb/c 3T3 cells synthesize 5–10 times more ³⁵SO₄^2?-labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular ³⁵SO₄^2?-labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70–80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65–75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular ³⁵SO₄^2?-labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 ³⁵SO₄^2?-labeled proteoglycans and contains chondroitin sulfate exclusively. It is altogether absent in the extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5–10-fold decreased synthesis of ³⁵SO₄^2?-labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42− uptake and expression of a band 3 like protein in the marine sponge,Microciona prolifera

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of ³⁵SO₄^2? uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in ³⁵SO₄^2? uptake and incorporation which could be greatly augmented in the presence of Ca²⁺/Mg²⁺. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa ³⁵SO₄^2? sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.     

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.     

Increased Retinal Synthesis of Heparan Sulfate Proteoglycan and HNK-1 Glycoproteins Following Photoreceptor Degeneration

Abstract: In an earlier analysis of the retinal biosynthesis of proteoglycan, we noted that, following photoreceptor degeneration in the rd (retinal degeneration) mouse, the remaining inner retina exhibited a marked elevation in synthesis of heparan sulfate proteoglycan (HSPG), well above the level observed in the normal (nondegenerate) retina, as well as a pronounced increase in sulfation of protein substrates. Biochemical and autoradiographic results of ³⁵S-amino acid utilization reported here confirm that the ³⁵SO₄^2? differences seen previously are accompanied by increased protein synthesis in the rd retina. An intact photoreceptor cell layer is neither a barrier to nor a sink for the amino acid precursor. Further, we have examined sulfate utilization in four other rodent strains with photoreceptor degenerations. In each of the models examined, an increase in retinal synthesis of ³⁵SO₄^2?-labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na₂³⁵SO₄ isolated retinal cultures from the following: (a) mice with light-induced photoreceptor degeneration; (b) rd mice; (c) transgenic mice with photoreceptor degeneration; (d) RCS rats; and (e) rats with light-induced photoreceptor degeneration. Comparisons were made with concurrent cultures of control nondegenerate retinal tissues. Protein and proteoglycan-enriched fractions were prepared from the incubation media and guanidine HCI/detergent extracts of the retinas by ion-exchange chromatography. The ³⁵SO₄^2?-proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of ³⁵SO₄^2?-HSPG found in control incubations. The RCS and light-damaged rats also showed increased synthesis of ³⁵SO₄^2?-chondroitin sulfate proteoglycan relative to the control, though the increase was of lesser magnitude than the HSPG effect. ³⁵SO₄^2?-protein in degenerate and light-damaged retinas always contained at least twice the radioactivity found in comparable control preparations. The bulk of the increased radiolabeling was found in N-linked oligosaccharides, including several recognized by the HNK-1 antibody. These data suggest that a sustained increase in HSPG and HNK-1 glycoprotein synthesis is a consistent response of inner retinal cells following loss of photoreceptors and is independent of the cause of photoreceptor degeneration.     

Sulfur dynamics in mineral horizons of two northern hardwood soils. A column study with 35S

Sulfur dynamics of two Spodosols were ascertained using soil columns constructed from homogenized mineral soil from nothern hardwood ecosystems at the Huntington Forest (HF) in the Adirondack Mountains of New York and Bear Brook Watershed in Maine (BBWM). Columns were leached for 20 weeks with a simulated throughfall solution with³⁵SO₄ ^2-. Sulfur constituents were similar to those of other Spodosols, with the organic S fractions (C-bonded S and ester sulfate) constituting over 90% of total S. HF soil columns had higher total S (14.9 mol S g^-1) than that for the BBWM soil columns (7.4 mol g^-1) primarily due to higher C-bonded S in the former.Initially, adsorbed SO₄ ^- accounted for 5 and 4% of total S for the BBWM and HF soil columns, respectively. After 20 weeks, adsorbed SO₄ ^2- decreased (81%) in BBWM and increased (33%) in HF soil columns. For both HF and BBWM soil columns, C-bonded S increased and ester sulfate decreased, but only for HF columns was there a net mineralization of organic S (5.6% of total S). The greatest decrease in ester sulfate occurred at the top of the columns.Leaching of³⁵S was less than 0.5% of the³⁵S added due to its retention in various S constituents. There was an exponential decrease in³⁵S with column depth and most of the radioisotope was found in C-bonded S (70–88 and 70–91% for BBWM and HF, respectively). The rapid turnover of adsorbed SO^2- ₄ was reflected in its high specific activity (834 and 26 kBq mol^-1 S for BBWM and HF, respectively). The lower specific activity of adsorbed SO₄ ^2- in HF was attributable to greater isotopic dilution by non-radioactive SO^2- ₄ derived from greater organic S mineralization in the HF versus the BBWM columns.Both soil columns initially had high levels of NO^- ₃ which resulted in the generation of H⁺ and net retention of SO₄ ^2- in the early phase of the experiment due to pH dependent sulfate adsorption; later NO₃ ^- decreased and SO₄ ^2- was desorbed. Leaching of NIO₃ ^- and SO₄ ^2- was correlated with losses of Mg²⁺ and Ca²⁺ of which the latter was the dominant cation.Analyses using both S mass balances and radioisotopes corroborate that for BBWM soil columns, SO^2- ₄ adsorption-desorption dominated the S biogeochemistry while in HF soil columns, organic S mineralization-immobilization processes were more important. It is suggested that similar techniques can be applied to soils in the field to ascertain the relative importances of SO₄ ^2- adsorption processes and organic S dynamics.     

Distribution of inorganic sulfate between plasma, liver, and bile

The distribution of ³⁵SO₄⁼ between inorganic and other forms in plasma, liver and bile of rats was studied by use of paper electrophoresis. The overall liver sulfate space varied from 50–100% but when determined for inorganic ³⁵SO₄⁼ was constant at 34%. This is considerably higher than that expected for passive distribution and suggests SO₄⁼ is actively transported into liver cells. Overall bile/plasma ratios were always greater than 1 while the ratio for inorganic sulfate was 0.44, a value consistent with the known bile/plasma electrical potential.     

Bioleaching of Heavy Metal Polluted Sediment: Kinetics of Leaching and Microbial Sulfur Oxidation

Remediation of heavy metal polluted sediment through bioleaching using elemental sulfur (S⁰) as the leaching agent can be regarded as a two‐step process: firstly, the microbial oxidation of the added S⁰ to sulfuric acid and, secondly, the reaction of the produced acid with the sediment. Here, both subprocesses were studied in detail independently: oxidized river sediment was either suspended in sulfuric acid of various strengths, or mixed with various amounts of finely ground S⁰ powder (diameter of the S⁰ particles between 1 and 175 μm with a Rosin‐Rammler‐Sperling‐Bennet (RRSB) distribution and an average diameter of 35 μm) and suspended in water. The leaching process was observed by repeated analysis of the suspension concerning pH, soluble sulfate and metals, and remaining S⁰. In the case of abiotic leaching with H₂SO₄, the reaction between the acid and the sediment resulted in a gradual increase in pH and a solubilization of sediment‐borne heavy metals which required some time; 80 % of the finally solubilized heavy metals was dissolved after 1 h, 90 % after 10 h, and 100 % after 100 h. In the case of bioleaching, the rate of S⁰ oxidation was maximal at the beginning, gradually diminished with time, and was proportional to the initial amount of S⁰. Due to its very low solubility in water, S⁰ is oxidized in a surface reaction catalyzed by attached bacteria. The oxidation let the particles shrink, their surface became smaller and, thus, the S⁰ oxidation rate gradually decreased. The shrinking rate was time‐invariant and, at 30 °C, amounted to 0.5 μm/day (or 100 μg/cm²/day). Within 21 days, 90 % of the applied S⁰ was oxidized. Three models with a different degree of complexity have been developed that describe this S⁰ oxidation, assuming S⁰ particles of uniform size (I), using a measured particle size distribution (II), or applying an adapted RRSB distribution (III). Model I deviated slightly from the measured data but was easy to handle, Model II fitted the measured data best but its simulation was complicated, and Model III was intermediate. The amount of soluble sulfate was smaller than the amount of H₂SO₄ added or microbially generated as the H₂SO₄ reacted with the sediment to form in part poorly soluble sulfates. A model has been developed that describes the pH and the soluble sulfate and metals at equilibrium, depending on the amount of H₂SO₄ applied or microbially generated, and that is based on the condition of electrical neutrality, a global metal/proton exchange reaction, and a sulfate‐fixation reaction. In suspension, bioleaching with S⁰ required considerably more time than abiotic leaching with H₂SO₄, but the final pH and metal solubilization were identical when equimolar amounts of leaching agents were applied.     

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.     

Cell surface proteoglycans as a negative modulator in concanavalin a-mediated agglutination of hepatoma cells

Proteoglycan (heparan sulfate-protein conjugate) was solubilized with 8 M urea from rat liver plasma membranes after enzymic (RNAase, neuraminidase) treatments and extensively purified by chromatography and gel filtration. The final products gave an average ratio of hexuronate to protein (weight) of approx. 1.5, contained hexosamine equimolar to hexuronate and were sensitive to β-elimination (the molecular weight being reduced from 20 · 10⁴ to 3 · 10⁴ (gel filtration)).The proteoglycan fraction, when added to trypsinized and untrypsinized ascites hepatoma (AH-130F(N)) cells, inhibited the concanavalin A-mediated agglutination of the cells. However, the alkali-treated proteoglycan (β-elimination) or acid mucopolysaccharide fraction prepared from liver plasma membranes by papain digestion were less effective, and a reference preparation of heparan sulfate was almost ineffective. It was confirmed that significant amounts of proteoglycan labelled with ³⁵SO₄^2? were firmly bound to or taken up by the trypsinized ascites hepatoma cells.These results together with the sensitization of lectin-mediated agglutination by mild protease treatment of cells suggest that cell surface proteoglycans may act as a negative modulator in the lectin-mediated agglutination of cells.     

Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L

The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty-four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH₄⁺ added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH₄⁺, the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH₄⁺ from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO₃⁻ as a nitrogen source. Using [³⁵S]SO₄²⁻ as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH₄⁺ was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH₄⁺ may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis.     

The Characterization of a Cerebroside Sulfate from the Mycelia of Glomerella cingulata

A sulfur-containing lipid with chromatographic properties in several systems equal to the commercial sulfolipid (extracted from bovine spinal cord, Applied Science) has been isolated in 0.6 % yield (dry weight basis) from the mycelia of Glomerella cingulata. About 35 % of exogenous Na³⁵SO₄ incubated with the mycelial medium for 72 hours was recovered in the cerebroside sulfate fraction, with only traces of ³⁵S activities in other lipid fractions.     

Kinetics of nitrate and sulfate removal using a mixed microbial culture with or without limited-oxygen fed

The biological degradation of nitrate and sulfate was investigated using a mixed microbial culture and lactate as the carbon source, with or without limited-oxygen fed. It was found that sulfate reduction was slightly inhibited by nitrate, since after nitrate depletion the sulfate reduction rate increased from 0.37 mg SO₄ ^2?/mg VSS d to 0.71 mg SO₄ ^2?/mg VSS d, and the maximum rate of sulfate reduction in the presence of nitrate corresponded to 56 % of the non-inhibited sulfate reduction rate determined after nitrate depleted. However, simultaneous but not sequential reduction of both oxy-anions was observed in this study, unlike some literature reports in which sulfate reduction starts only after depletion of nitrate, and this case might be due to the fact that lactate was always kept above the limiting conditions. At limited oxygen, the inhibited effect on sulfate reduction by nitrate was relieved, and the sulfate reduction rate seemed relatively higher than that obtained without limited-oxygen fed, whereas kept almost constant (0.86–0.89 mg SO₄ ^2?/mg VSS d) cross the six R_OS states. In contrast, nitrate reduction rates decreased substantially with the increase in the initial limited-oxygen fed, showing an inhibited effect on nitrate reduction by oxygen. Kinetic parameters determined for the mixed microbial culture showed that the maximum specific sulfate utilization rate obtained (0.098?±?0.022 mg SO₄ ^2?/(mg VSS h)) was similar to the reported typical value (0.1 mg SO₄ ^2?/(mg VSS h)), also indicating a moderate inhibited effect by nitrate.     

Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice

Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [³⁵S]-PAPS only or [³⁵S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H₂³⁵SO₄. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. $\text{et}\text{al}$. (1976) Devel. Biol. $\text{50}$, 82–94) may result from a defect in the synthesis of the sulfate donor PAPS.     

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte

Equilibrium exchange of SO₄^2? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO₄^2? was measured by both efflux and influx of ³⁵SO₄^2?. The rate of influx of SO₄^2? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO₄^2? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO₄^2? may occur through a DIDS-insensitive pathway.     

Biosynthesis of protein by microscopic fungi in solid state fermentation. II. Optimization of aspergillus oryzae A. or. 11 cultivation for protein enrichment of starchy raw materials

Study was made of the effect of medium humidity, source and dose of nitrogen, dose of phosphorus, dose of inoculum and aeration on protein biosynthesis by strain Aspergillus oryzae A. or. 11 in solid state fermentation. It was found that for a maximal protein yield the medium ought to contain about: 60% of water, 36% of starchy raw materials d. m. (e.g. 28.8% of coarse rye meal and 7.2% of beet pulp), 0.85% of nitrogen sources (1.6% of (NH₄)₂SO₄ and 1.1% of urea), and 0.35% of phosphorus source (1.3% of KH₂PO₄). pH of medium should be near 6.5. The dose of inoculum should not be lower than 10⁸ spores/100 g medium. The duration of culture ought to be 20–22 h at 30–35°C, with aeration at least 40 dm³ of air/h × 1000 g medium. Under these conditions the protein yield is about 6.0–6.3 g/100 g of starting medium d.m. at the cost of utilization of about 25 g total carbohydrates.     

Sulfate flux in high sodium cat red cells

The transport of radioactive sulfate in cat red cells has been studied. The rate constant for ³⁵SO₄ inward movement under steady-state conditions is 0.24 ± 0.02/hr. This movement was found to be sensitive to osmotic changes in cell volume and to the nature of anions in the incubation medium; it increases with increasing cell volume and decreases with decreasing cell volume. The anions SCN, NO₃, and I were found to inhibit the uptake of ³⁵SO₄. Furthermore, 1-fluoro-2,4-dinitrobenzene at a concentration of 1 mM inhibits (>90%) this uptake. The inward movement of erythritol-¹⁴C shows qualitatively the same dependence on cell volume as ³⁵SO₄, but it is insensitive to the nature of the anion present in the bathing medium. It was also found that the usually observed inhibition of radioactive Na uptake by SCN in cat red cells can be reversed when cell volume is increased.     

Heparan sulfate at the surface of HeLa cells

Summary HeLa cells, labeled with Na₂ ³⁵SO₄, release into the culture medium³⁵SO₄ bound to plasma membrane vesicles next to³⁵SO₄-glycoproteins and free³⁵SO₄. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain³⁵SO₄ and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.Supported by a grant from the Algemene Spaar- en Lijfrentekas Cancer Fund, Brussels, Belgium.     

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?.     

Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter

Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [³⁵S]SO₄²⁻ accumulation. Maximum uptake is secured in the presence of Mg²⁺ and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [³⁵S]SO₄²⁻ uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter.     

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH₄)₂SO₄], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH₄)₂SO₄ system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH₄)₂SO₄ and 1.0?mol l^?1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.