Assimilatory Sulfur Metabolism in Marine Microorganisms: Considerations for the Application of Sulfate Incorporation into Protein as a Measurement of Natural Population Protein Synthesis

The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [³⁵S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of ³⁵S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [³⁵S]sulfate incorporation was compared with [¹⁴C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with ³⁵S. Redistribution of ¹⁴C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [³⁵S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.     

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.     

Kinetics of Sulfate and Acetate Uptake by Desulfobacter postgatei

The kinetics of sulfate and acetate uptake was studied in the sulfate-reducing bacterium Desulfobacter postgatei (DSM 2034). Kinetic parameters (K_m and V_max) were estimated from substrate consumption curves by resting cell suspensions with [³⁵S]sulfate and [¹⁴C]acetate. Both sulfate and acetate consumption followed Michaelis-Menten saturation kinetics. The half-saturation constant (K_m) for acetate uptake was 70 μM with cells from either long-term sulfate- or long-term acetate-limited chemostat cultures. The average K_m value for sulfate uptake by D. postgatei was about 200 μM. K_m values for sulfate uptake did not differ significantly when determined with cells derived either from batch cultures or sulfate- or acetate-limited chemostat cultures. Acetate consumption was observed at acetate concentrations of ≤1 μM, whereas sulfate uptake usually ceased at 5 to 20 μM. The results show that D. postgatei is not freely permeable to sulfate ions and further indicate that sulfate uptake is an energy-requiring process.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Technique for Simultaneous Determination of [35S]Sulfide and [14C]Carbon Dioxide in Anaerobic Aqueous Samples

A technique for the simultaneous determination of [³⁵S]sulfide and [¹⁴C]carbon dioxide produced in anaerobic aqueous samples dual-labeled with [³⁵S]sulfate and a ¹⁴C-organic substrate is described. The method involves the passive distillation of sulfide and carbon dioxide from an acidified water sample and their subsequent separation by selective chemical absorption. The recovery of sulfide was 93% for amounts ranging from 0.35 to 50 μmol; recovery of carbon dioxide was 99% in amounts up to 20 μmol. Within these delineated ranges of total sulfide and carbon dioxide, 1 nmol of [³⁵S]sulfide and 7.5 nmol of [¹⁴C]carbon dioxide were separated and quantified. Correction factors were formulated for low levels of radioisotopic cross-contamination by sulfide, carbon dioxide, and volatile organic acids. The overall standard error of the method was ±4% for sulfide and ±6% for carbon dioxide.     

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.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

Some observations on the biosynthesis of the plant sulpholipid by Euglena gracilis

1. dl-Cysteine decreases the uptake of ³⁵SO₄²⁻ by Euglena gracilis but does not decrease the relative incorporation of the isotope into sulpholipid; cysteic acid, on the other hand, does not affect the uptake of ³⁵SO₄²⁻ but does dilute out its incorporation into the sulpholipid. 2. Both l-[³⁵S]cysteic acid and dl-+meso-[3-¹⁴C]cysteic acid appear almost exclusively in 6-sulphoquinovose. 3. Molybdate inhibits the incorporation of ³⁵SO₄²⁻ into sulpholipid but not its uptake into the cells; this suggests that adenosine 3′-phosphate 5′-sulphatophosphate may be concerned with the biosynthesis of sulpholipid, and it was shown to be formed by chloroplast fragments. 4. An outline scheme for sulpholipid biosynthesis based on these observations is discussed.     

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.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs: I. Chlorfluazuron,diflubenzuron, and teflubenzuron inhibit incorporation but not uptake of [14C]N-acetyl-D-glucosamine

The purpose of this study was to determine the effects of potent inhibitors of chitin synthesis on an organ culture test system as a basis for determining the mode of action of such compounds. Consequently, we investigated the action of chlorfluazuron (CFA), diflubenzuron (DFB), and teflubenzuron (TFB) on uptake and incorporation into chitin of [¹⁴C]N-acetyl-D-glucosamine ([¹⁴C]GlcNAc) in wing imaginal discs cultured in vitro. Spodoptera frugiperda wing imaginal discs provided a highly responsive test system for studying the inhibition of ecdysteroid-dependent chitin synthesis in a target tissue in vitro. All three inhibitors blocked ecdysteroid-dependent [¹⁴C]GlcNAc incorporation into chitin by the wing imaginal discs. The effectiveness of the inhibitors was not affected by the time of their application, i.e., exposures before, during, or after 20-hydroxyecdysone treatment were equally effective in inhibiting chitin synthesis. Thus, exposure of freshly dissected discs to CFA for periods as short as 15 min inhibited approximately 90% of the chitin synthesis measured 72 h later. In contrast to previous in vivo studies all three inhibitors were similar in their effectiveness in vitro. However, while all three compounds inhibited [¹⁴C]GlcNAc incorporation in a similar dose-dependent manner, only DFB and TFB reduced but did not block uptake of GlcNAc. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Simple Assay for Accurate Determination of [35S]sulfate Reduction Activity

A method is described for the assay of [³⁵S]sulfate reduction in which filter paper wicks are used to trap [³⁵S]sulfide. The simplicity of the technique enables large numbers of samples to be conveniently processed. Enhanced sensitivity is achieved since all acid-volatile [³⁵S]sulfides produced during the incubation period are counted. Recovery of radioactivity from added Na₂³⁵S is excellent (mean, 100.1%; standard deviation, 1.81; n = 9) and is unaffected by sulfide concentrations of up to 400 μg per sample. Field trial results with anoxic sediment samples are presented.     

Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins

A bioassay to measure the incorporation of [¹⁴C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [¹⁴C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [¹⁴C]leucine uptake. The effectiveness of the wall fragments in inhibiting [¹⁴C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.     

Endosperm Protein Synthesis and l-[S]Methionine Incorporation in Maize Kernels Cultured In Vitro

This study was conducted to examine protein synthesis and l-[³⁵S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[³⁵S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.     

Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthesis.

The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in vitro. At levels of 10^?9 to 10^?8m retinoic acid, ³⁵SO₄ uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of ³⁵SO₄ or [¹⁴C]glucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased three- to fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin 6-sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of ³⁵SO₄ into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in cultured fibroblasts

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/μg DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/μg DNA). After incubation of fibroblasts with [³H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [³⁵S]sulfate resulted in the incorporation of [³⁵S]sulfate into 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [³⁵S]sulfate and [³H]glucosamine from 5 min to 16 h showed that [³⁵S]PAPS was equilibrated in less than 10 min, while [³H]glucosamine required a longer time, 2–4 h, to attain a steady state with UDP-N-acetylhexosamine. [¹⁴C]Glucose required approximately the same time as [³H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.