Endothelial heparan sulphate: Compositional analysis and comparison of chains from different proteoglycan populations

From cultures of human umbilical vein endothelial cells incubated with³H-glucosamine or³⁵S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (>1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW>50,000) of low overall sulphation (32–35%N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.Abbreviations HSPG heparan sulphate proteoglycan - DSPG dermatan sulphate proteoglycan - GlcNAc(6S) N-acetylglucosamine 6-sulphate - GlcNAc6R glucosamine with either-OH or-OSO₃ at C-6 - GlcNR glucosamine with either-SO₃ or-COCH₃ as N-substituent - GlcNSO₃ N-sulphated glucosamine - GlcNSO₃(3S) N-sulphated glucosamine 3-sulphate - GlcA d-glucuronic acid - IdoA l-iduronic acid - IdoA(2S) iduronic acid 2-sulphate - HexA hexuronic acid - DHexA hexuronic acid with a 4,5-double bond - Xyl xylose - SAX strong anion exchange - d.p. degree of polymerization (a disaccharide has d.p.=1 etc) - AUFS absorbance units full scale The codes used for proteoglycans denote in turn: C 2, low-density (1.35–1.28 g/ml) HSPG from the cell extract; M 1a, high density (>1.4 g/ml) HSPG fraction from the spent medium; M 2a, low-density (1.31 g/ml) HSPG from the spent medium [6].     

Peptidoglycan Loss During Hen Egg White Lysozyme-Inorganic Salt Lysis of Streptococcus mutans

Streptococcus mutans BHT was grown in Todd-Hewitt dialysate medium containing N-acetyl[¹⁴C]glucosamine for 6 to 11 generations. After treatment with cold and hot trichloroacetic acid and trypsin, 52 to 65% of the radioactivity remained present in insoluble peptidoglycan-containing residues. Hen egg white lysozyme or mutanolysin treatment of the peptidoglycan residues resulted in the release of 80 and 97%, respectively, of the ¹⁴C label to the supernatant fraction. Hydrochloric acid hydrolysates of such supernatants showed that essentially all of the radioactivity present in insoluble peptidoglycan fractions was present in compounds that comigrated on paper chromatography with glucosamine (~60%) or muramic acid (~30%). Treatment of whole cells with low and high concentrations of lysozyme alone resulted in losses of 45 and 70% of the insoluble peptidoglycan, respectively, yet release of deoxyribonucleic acid from cells was not detected. Sequential addition of appropriate concentrations of selected inorganic salts after lysozyme treatment did result in the liberation of deoxyribonucleic acid. Deoxyribonucleic acid release was correlated with a further release of peptidoglycan from the insoluble fraction. However, the total amount of peptidoglycan lost effected by the low concentration of lysozyme and NaSCN (lysis) was significantly less than the amount of peptidoglycan hydrolyzed by high concentrations of lysozyme alone (no lysis), suggesting that the overall amount of peptidoglycan lost did not correlate well with cellular lysis. The total amount of insoluble peptidoglycan lost at the highest salt concentrations tested was found to be greater than could be accounted for by lysozyme-sensitive linkages of the peptidoglycan, possibly implicating autolysins. The results obtained suggested that hydrolysis of peptidoglycan bonds in topologically localized, but strategically important, sites was a more significant factor in the sequence that results in loss of cellular integrity (lysis).     

Effect of Penicillium chrysogenum on Lignin Transformation

A strain of Penicillium chrysogenum has been isolated from pine forest soils in Tenerife (Canary Islands). This strain was capable of utilizing hydroxylated and nonhydroxylated aromatic compounds, in particular cinnamic acid, as its sole carbon source. In an optimum medium with high levels of nitrogen (25.6 mM) and low levels of glucose (5.5 mM), it was able to decolorize Poly B-411 and to transform kraft, organosolv, and synthetic dehydrogenative polymerisate lignins. After 30 days of incubation, the amount of recovered kraft lignin was reduced to 83.5 and 91.3% of that estimated for uninoculated controls by spectrophotometry and klason lignin, respectively. At the same time, the pattern of molecular mass distribution of the lignin remaining in cultures was changed. The amount of organosolv lignin recovered from cultures was reduced to 90.1 and 94.6% of the initial amount as evaluated by spectrophotometry and klason lignin, respectively. About 6% of total applied radioactivity of O¹⁴CH₃-organosolv lignin was recovered as ¹⁴CO₂ after 30 days of incubation, and 18.5% of radioactivity from insoluble O¹⁴CH₃-organosolv lignin was solubilized. After 26 days of incubation, 2.9% of ¹⁴C-β-dehydrogenative polymerisate and 4.1% of ¹⁴C-ring-dehydrogenative polymerisate evolved as ¹⁴CO₂.     

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.     

Biosynthesis of biopterin in Ascaris lumbricoides suum

In in vivo experiments, radioactivity from [U-¹⁴C]GTP was incorporated into biopterin, and, in fact, all carbon atoms of biopterin synthesized in Ascaris lu lumbricoides suum originated from GTP.Biopterin was also biosynthesized in homogenates of tissue fluid and muscles of Ascaris lumbricoides suum.The enzyme which catalyzes sepiapterin synthesis from D-erythto-7,8-dihydroneopterin-3′-phosphate was found in A. lumbricoides suum extracts and extracted in the 0–30% (NH₄)₂SO₄ fraction from a 40 000 × g supernatant. The enzyme was purified by Sephadex G-200 column and DEAE-cellulose column chromatography. It is suggested that sepiapterin could be an intermediate compound in biopterin biosynthesis.     

Extracellular Isoamylase Produced by the Yeast Lipomyces kononenkoae

A strain of the starch-converting yeast Lipomyces kononenkoae produced, when grown on starch, a debranching enzyme that proved to be an isoamylase (glycogen 6-glucanohydrolase; E.C. 3.2.1.68). So far, only bacteria have been found to produce extracellular isoamylases. The yeast isoamylase enhanced β-amylolysis of amylopectin and glycogen and completely hydrolyzed these substrates into maltose when combined with a β-amylase but had no action on dextran or pullulan. By isopropanol precipitation and carboxymethyl cellulose chromatography, L. kononenkoae isoamylase was partially purified from the supernatant of cultures grown on a mineral medium with soluble starch. Optimum temperature and pH for activity of the isoamylase were 30°C and 5.6. The molecular weight was around 65,000, and the pI was at pH 4.7 to 4.8. The K_m (30°C, pH 5.5) for soluble starch was 9 g liter⁻¹.     

Localization of hyaluronic acid in synovial cells by radioautography

Cultured human synovial cells secrete hyaluronic acid (HA) into the culture medium. Glucosamine-6-³H was shown to be a direct and relatively specific precursor of HA-³H by the following observations: the susceptibility of nondialyzable radioactivity in the medium to hyaluronidase, its migration with hexuronic acid on zone electrophoresis in polyvinyl chloride, its exclusion from Sephadex G-200, and the localization of radioactivity to glucosamine after hydrolysis of the labeled polysaccharide. The presence of intracellular HA-³H was established by sequential extraction of labeled cells and by radioautography of synovial cell cultures digested with hyaluronidase in situ. When cells were exposed to medium lacking glucose, glucosamine-³H-uptake was enhanced; and this made possible electron microscopic radioautographic studies. These studies demonstrate the early and continued presence of HA-³H within the Golgi apparatus.     

Biosynthesis of chondroitin sulfate side chains and hyaluronate time-course studies with cartilage slices of calf ribs under different conditions

The time course of double labeling with ³⁵SO₄²⁻ and [³H]glucosamine was followed in a semi-in vitro system of cartilage slices from calf ribs whose chondroitin sulfate peptide pool consistsof (A) <1% of very short undersulfated side chains of <10 disaccharide units length, (B) 3–5% of short undersulfated longer side chains (16 to 25 disaccharide units), (C) 3–5% of short, slightly oversulfated side chains (16–23 dissacharide units, very probably containing some dermatan sulfate), (D) the bulk material (74–82% of total uronate) of longest, slightly undersulfated or equally sulfated side chains (22–42 disaccharide units).After 10 min incubation rapid chain elongation with [³H]glucosamine and prelabeling with ³⁵SO₄²⁻ of endogenous acceptors are apparent. Chains of type A exhibit highest specific radioactivities. During 30–60 min incubation it is mainly chains of type B that show highest specific radioactivities, after 90 min chains of type C. On the after hand, chains of type D always incorporated the highest total amount of both precursors. Preincubation of slices for 40 min at 37°C strongly enhances labeling rates of all types whilst preincubation for 40 min in an ice-bath enhances mainly ³⁵SO₄²⁻ labeling of types A and B.After 10 min preincubation followed by ³⁵SO₄²⁻ labeling for 60 min a decrease of radioactivity of type A and a distinct increase with type B are observed during the post incubation period. After pulse chase experiment type B exhibit highest specific radioactivities. The data make it evident that under-sulfated short chondroitin sulfate side chains from very rapidly in a well organised manner and grow, by elongation and proceeding sulfation processes, to longer higher sulfated chains.The labeling of the hyaluronate pool is about half of that of the chondroitin sulfate pool after a lag phase of 10 min. The latter increases linearly after 35–45 min incubation time. However, after preincubation and chase experiments the hyaluronate pool is more highly labeled. The data indicate different precursor pools of both biosynthesis mechanisms, probably located in different cell compartments and/or different cartilage cells.     

Inhibition of lymphoma cell proliferation by supernatant from fibrosarcoma cultures: Preliminary evidence that the inhibitory material is prostaglandin E

Supernatants obtained from mouse fibrosarcoma cultures 48 hr after the addition of fresh medium contained dialyzable material which inhibited the proliferation of syngeneic lymphoma cells , as measured by ³H-thymidine incorporation. Three lines of evidence indicate that the supernatant inhibitory material is probably prostaglandin (PG) E. First, the supernatant and dialysate of the supernatant contained a substance with the same characteristics as PGE₁ or PGE₂ as detected by thin layer chromatography. Second, PGE₂-treatment of lymphoma cells mimicked the inhibition of proliferation observed with supernatant inhibitory substance. Third, indomethacin treatment of fibrosarcoma cultures reduced the amount of supernatant inhibitory substance present.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

The purification and study of a honey bee abdominal sucrase exhibiting unusual solubility and kinetic properties.

A sucrase from honey bee abdomens was purified to a high state of homogeneity. It was unusual in that it was completely soluble in high concentrations of ammonium sulfate and because curved rather than rectilinear lines were obtained when initial velocity data for at least two substrates were plotted. The action of the enzyme towards a large number of glycosides showed that the enzyme was able to hydrolyze all α-glucosides tested except trehalose and starch. pH Optima of sucrose and p-nitrophenyl-α-d-glucopyranoside differed by 1.0 pH unit. The unusual kinetic patterns which were found seem to be unique to this disaccharidase and were shown to be the result of a combination of hydrolytic and transferolytic activity in which the initial substrate is also a very good acceptor molecule for the transferolytic process. The K_m value for hydrolysis was found to be about an order of magnitude lower than for other insect sucrases with the more usual type of kinetic action. Amino acid and amino sugar analyses showed that the sucrase was a glycoprotein which contained glucosamine and either mannosamine or galactosamine. The molecular weight of the enzyme was estimated to be 70,000 or higher and there was no evidence that the enzyme had subunit structure. An s⁰_20,w value of 5.3S was determined. The enzyme was quite stable to a series of denaturing conditions and sulfhydryl reacting agents had little effect on the activity.     

Production of Manganese Peroxidase and Organic Acids and Mineralization of 14C-Labelled Lignin (14C-DHP) during Solid-State Fermentation of Wheat Straw with the White Rot Fungus Nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic ¹⁴C-ring-labelled lignin (¹⁴C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as ¹⁴CO₂, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the ¹⁴C-DHP to ¹⁴CO₂ (which accounted for up to 8% of the initial radioactivity added) and ¹⁴C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

Inhibition of globin chain initiation in reticulocyte lysates by pactamycin: accumulation of methionyl-valine

In a globin synthesizing system containing reticulocyte lysate and [³⁵S] met-tRNA_f, low levels of pactamycin cause an accumulation of radioactivity on the monosomes and small oligosomes concomitant with the breakdown of polysomes. About 50% of the ribosome-bound radioactivity corresponds to methionyl-valine, the initial dipeptide of globin chains, with negligible amounts of tri- or other oligopeptides. This suggests that the site of its action is after the formation of the first peptide bond. The ribosome-bound radioactivity in the presence of sparsomycin, an inhibitor of chain elongation, is in di-, tri- and oligopeptides. Sparsomycin levels (10^?5M) that cause almost complete inhibition of polypeptide synthesis have only a small inhibitory effect on the pactamycin-induced accumulation of methionyl-valine. Fusidic acid and chlortetracycline do not cause accumulation of any significant amounts of methionyl-valine.     

Tumor necrosis factor reduces proteoglycan synthesis in cultured endothelial cells

Tumor necrosis factor (TNF)-induced disruption of vascular endothelial barrier function may be due in part to alterations in proteoglycan metabolism. To test this hypothesis, confluent endothelial cell monolayers were exposed for 24 h to 500 or 1,000 U of TNF per mililiter of culture medium together with 20 μCi Na₂ ³⁵SO₄. HPLC anion-exchange separation of proteoglycans secreted into media of control as well as TNF-treated cultures revealed one major peak (representing 95% of total radioactivity) and one minor peak (representing 5% of total radioactivity), which eluted at 0.6 and 0.9 M NaCl, respectively. One single peak was obtained from control as well as TNF-treated endothelial cell monolayers and eluted at 1.2 M NaCl. TNF treatment did not change the total quantity of radioactive proteoglycans secreted into the media but significantly decreased the amount of proteoglycans in endothelial cell monolayers. However, TNF treatment did not alter the size or glycosaminoglycan (GAG) composition of the proteoglycans either in the media or in the cell monolayers. In addition, mRNA levels of specific proteoglycans, perlecan and biglycan, were measured upon TNF treatment, using Northern analysis. TNF treatment caused a dose-dependent decrease in mRNA levels for the biglycan in endothelial cultures. These results suggest that TNF decreases production of proteoglycans and alters normal endothelial cell proteoglycan metabolism which may be sufficient to impair endothelial barrier function. © 1995 Wiley-Liss, Inc.     

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6¹,6³- and 6¹,6⁴-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the k_cat and K_m values for transglycosylation were 1.78 × 10³ s⁻¹ and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10³ s⁻¹ and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the k_cat/K_m ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.     

Breakdown of cartilage proteoglycan in a tissue culture model of rheumatoid arthritis

Proteoglycan breakdown was studied in a coculture model which mimics the confrontation between synovium and cartilage that occurs in rheumatoid arthritis. Bovine nasal-septum cartilage discs radioactively labeled (³⁵SO₄^2? with or without [³H]glucosamine) and ‘chased’ in non-radioactive medium were cultured in contact with minced rheumatoid synovial membranes for intervals up to 8 days. Synovium-stimulated (2–3 fold) cartilage breakdown was unaffected by ascorbate supplementation. Labeled products (small molecules plus proteoglycan complexes) in culture media were characterized by chromatographic, sedimentation and enzymic digestion methods. Breakdown was dominated by the release of a range of proteoglycan products, fully disaggregated and incapable of reaggregation with added hyaluronate. Because constituent glycosaminoglycans were of uniform size, proteoglycan polydispersity was attributed to differences in core protein length. Hydrocortisone inhibited degradation and partially prevented the shift of proteoglycans to lower average molecular weight. An additional breakdown pattern occasionally noted during the initial 48 h of coculture was characterized by release of a subpopulation of low charge-density proteoglycan bearing shortened glycosaminoglycan chains, consistent with glycosidase action. We conclude that rheumatoid synovia exhibit two distinct cartilage degradative potencies in vitro that may be important in vivo: (a) A variable hyaluronidase-like activity at early culture times, and (b) a dominant proteolytic activity generating an array of disaggregated proteoglycann products that differ largely on the basis of their core lengths. The response to hydrocortisone is consistent with inhibition of proteolysis through the stabilization of cellular membranes.     

Synthesis and Release of Cyclic Adenosine 3':5'-Monophosphate by Ochromonas malhamensis

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3′:5′-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [³²P]cAMP was purified from cultures supplied [³²P]phosphate. The compound was identified as [³²P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a ³²P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a ³²P compound with the chromatographic behavior of 5′-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [³H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step.     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.     

Plant Glycoprotein biosynthesis Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root

A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-d-[¹⁴C₁]glucose from uridine diphosphate 2-acetamido-2-deoxy-d-[¹⁴C₁]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation.Hydrolysis of the reaction product yielded glucosamine as the only radiolabeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-d-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100°C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease.