scholarly journals The serum opacity reaction of Streptococcus pyogenes: general properties of the streptococcal factor and of the reaction in aged serum

1968 ◽  
Vol 66 (1) ◽  
pp. 37-47 ◽  
Author(s):  
M. J. Hill ◽  
Lewis W. Wannamaker
Keyword(s):  
Streptococcus Pyogenes ◽  
Divalent Cations ◽  
Horse Serum ◽  
Sodium Deoxycholate ◽  
Heat Stability ◽  
Maximal Activity ◽  
Free Enzyme ◽  
Ph Optimum ◽  
Serum Opacity Factor ◽  
High Concentrations

SUMMARYThe capacity of certain strains of Streptococcus pyogenes to produce opacity in aged horse serum has been studied. Cells from all stages of the growth cycle are able to produce opacity. Maximal activity is reached towards the end of the exponential phase of growth.Examination of cell fractions obtained by mechanical breakage and differential centrifugation suggested that the cell-bound activity is predominantly associated with the membrane fraction. Extraction with sodium deoxycholate yields a soluble fraction of high activity.There is considerable strain variation in heat stability of the serum opacity factor. Cell-bound activity is often quite resistant to heat, whereas extracted activity is less stable.Low concentrations of divalent cations have an activating effect, whereas high concentrations inhibit the serum opacity reaction. High concentrations of uni-valent cations are without effect on the cell-free enzyme but have an activating effect on the cell-bound enzyme.For both the cell-bound and the cell-free enzyme the pH optimum was 5·8.Although sensitive to trypsin and pepsin, the serum opacity factor appears to be resistant to streptococcal proteinase. Its activity is destroyed by formaldehyde and by periodate but is unaffected by a number of reducing agents.Pre-heating of the serum or the addition of iodoacetate did not affect the serum opacity reaction. The enhanced cholesterol esterification previously described with fresh serum appears to be a secondary reaction. Even when isolated by relatively gentle methods, α-lipoprotein serves as a substrate only in the presence of crystalline serum albumin.

Metabolism of phosphoglycerides in E. coli IV. The positional specificity and properties of phospbolipase A

1969 ◽  
Vol 47 (12) ◽  
pp. 1125-1128 ◽  
Author(s):  
P. Proulx ◽  
C. K. Fung
Keyword(s):  
Sodium Lauryl Sulfate ◽  
Divalent Cations ◽  
Sodium Deoxycholate ◽  
Maximal Activity ◽  
Phospholipase A ◽  
Lauryl Sulfate ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
E Coli

The hydrolysis of labelled phosphatidylethanolamine by E. coli was studied in vitro. Phospholipase A, as detected by 32P-labelled lysophosphatidylethanolamine formation, had two pH optima, 5 and 8.4. On the other hand lysophosphofipase was active only in the alkaline range, had a pH optimum of 10, and was inhibited by high concentrations of either sodium deoxycholate or sodium lauryl sulfate. Phospholipase A required Ca2+ addition for maximal activity at both pH optima. Mg2+ also stimulated the activity but other divalent cations tested were slightly inhibitory or without effect. Sodium lauryl sulfate completely inhibited at pH 5. Experiments with singly and doubly labelled phosphatidylethanolamine indicated that phospholipase A1 activity was predominant at both acid and alkaline pH. Lower levels of phospholipase A2 were detectable only at alkaline pH.

scholarly journals The Bacterium Thermus thermophilus, Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

2003 ◽  
Vol 69 (6) ◽  
pp. 3272-3279 ◽  
Author(s):  
Nuno Empadinhas ◽  
Luciana Albuquerque ◽  
Anke Henne ◽  
Helena Santos ◽  
Milton S. da Costa
Keyword(s):  
Biosynthetic Pathway ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximal Activity ◽  
Thermophilic Bacterium ◽  
Hyperthermophilic Archaea ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Homologous Genes ◽  
Molecular Masses

ABSTRACT The biosynthetic pathway for the synthesis of the compatible solute α-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of α-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90�C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95�C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn2+ was most effective for MPGS, while Mn2+, Co2+, Mg2+, and to a lesser extent Ni2+ activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

scholarly journals Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences

1998 ◽  
Vol 331 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Robert G. SPIRO ◽  
Vishnu D. BHOYROO
Keyword(s):  
Periodate Oxidation ◽  
Maximal Activity ◽  
Sialic Acid Residue ◽  
Lymphoid Tissues ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Selectin Ligands ◽  
Strict Requirement ◽  
Lectin Chromatography ◽  
Triton X

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.

STUDIES ON SOLUBILIZED ADENYLATE KINASE FROM MITOCHONDRIA

1966 ◽  
Vol 44 (11) ◽  
pp. 1469-1475 ◽  
Author(s):  
Marjorie A. Brewster ◽  
Ezzat S. Younathan
Keyword(s):  
Activation Energy ◽  
Rat Liver ◽  
Adenylate Kinase ◽  
Divalent Cations ◽  
Metabolic Function ◽  
Kcal Mole ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sensitive Method

Adenylate kinase from mitochondria of rat liver was made soluble by sonication. The enzyme had a pH optimum of 8.0, temperature optimum of 30°, and activation energy of 12.2 kcal/mole. It was activated by several divalent cations in the following order of efficiency: Mg++ > Co++ > Mn++ > Ca++, with an optimal Mg++: ADP ratio of 1. The apparent Km value (ADP as substrate) was found to be 1.3 mM at pH 7.4 and 30°. The activity was sensitive to phloretin and mildly activated by aurovertin. Oligomycin, 2,4-dinitrophenol, p-chloromercuribenzoate, alloxan, and phlorizin had no effect on the activity. The metabolic function and a comparison of the properties of this solubilized mitochondrial adenylate kinase with those of similar preparations from other sources are discussed in the light of these findings. During this study, a sensitive method adaptable for a large number of assays of adenylate kinase was developed, and is described in detail.

scholarly journals Genetic characterization of Streptococcus pyogenes emm 89 strains isolated in Japan from 2011 to 2019

2020 ◽  
Author(s):  
Yujiro Hirose ◽  
Masaya Yamaguchi ◽  
Norihiko Takemoto ◽  
Tohru Miyoshi-Akiyama ◽  
Tomoko Sumitomo ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Genetic Characterization ◽  
Streptococcal Toxic Shock Syndrome ◽  
Toxic Shock ◽  
Gene Encoding ◽  
Serum Opacity Factor ◽  
Genetic Features ◽  
Fibronectin Binding Protein

Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes emm 89 strains has been increasing in several countries and reported to be linked with a recently emerged clade of emm89 strains, designated clade 3. In Japan, epidemiological and genetic information for emm89 strains remains elusive. In this study, we utilized emm89 strains isolated from both STSS (89 isolates) and non-STSS (72 isolates) infections in Japan from 2011 to 2019, and conducted whole-genome sequencing and comparative analysis, which resulted in classification of a large majority into clade 3 regardless of disease severity. In addition, STSS-associated genes and SNPs were found in clade 3 strains, including mutations of streptokinase (Ska), control of virulence sensor (CovS), serum opacity factor (SOF), sortase (SrtB), and fibronectin-binding protein F1 (PrtF1), and absence of the hylP1 gene encoding hyaluronidase. These findings provide insights into notable genetic features of emm89 strains.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pisymport of the PiT family paralogs human PiT1 and PiT2

2006 ◽  
Vol 291 (6) ◽  
pp. C1377-C1387 ◽  
Author(s):  
Pernille Bøttger ◽  
Susanne E. Hede ◽  
Morten Grunnet ◽  
Boy Høyer ◽  
Dan A. Klærke ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Xenopus Laevis Oocytes ◽  
Transport Function ◽  
Knockout Mutant ◽  
Uptake Medium ◽  
The Impact ◽  
First Time

The general phosphate need in mammalian cells is accommodated by members of the Pitransport (PiT) family ( SLC20), which use either Na+or H+to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi(NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with32Pias a traceable Pisource. For PiT1, the Michaelis-Menten constant for Piwas determined as 322.5 ± 124.5 μM. PiT2 was analyzed for the first time and showed positive cooperativity in Piuptake with a half-maximal activity constant for Piof 163.5 ± 39.8 μM. PiT1- and PiT2-mediated Na+-dependent Piuptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+dependency patterns. However, only PiT2 was capable of Na+-independent Pitransport at acidic pH. Study of the impact of divalent cations Ca2+and Mg2+revealed that Ca2+was important, but not critical, for NaPitransport function of PiT proteins. To gain insight into the NaPicotransport function, we analyzed PiT2 and a PiT2 Pitransport knockout mutant using22Na+as a traceable Na+source. Na+was transported by PiT2 even without Piin the uptake medium and also when Pitransport function was knocked out. This is the first time decoupling of Pifrom Na+transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55and E575are responsible for linking Piimport to Na+transport in PiT2.

Characterization and distribution of arginine kinase in the tissues of the scorpion, Palamneus phipsoni

1985 ◽  
Vol 63 (10) ◽  
pp. 2262-2266 ◽  
Author(s):  
A. V. Arjunwadkar ◽  
S. Raghupathi Rami Reddy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Alimentary Canal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Arginine Kinase ◽  
High Specificity ◽  
Ph Optimum ◽  
High Concentrations ◽  
Claw Muscle ◽  
Slight Inhibition

Arginine kinase in claw muscle extracts of the scorpion, Palamneus phipsoni, was characterized. The enzyme, with a pH optimum of 8.5 in the direction of phosphoarginine synthesis, showed activation by Mg2+, high specificity towards L-arginine as the guanidino substrate, slight inhibition by high concentrations of L-arginine and ATP, and a molecular weight of 33 500. On polyacrylamide gel electrophoresis at pH 8.3 the enzyme migrated to the anode as a single molecular species. In addition to the claw muscle, the enzyme activity was also found to be present in the heart, alimentary canal, hepatopancreas, and nervous system. In general, scorpion muscle arginine kinase appears to be similar in its properties to the enzyme from other arthropods.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

1988 ◽  
Vol 66 (5) ◽  
pp. 425-435 ◽  
Author(s):  
Amy Mok ◽  
Tanya Wong ◽  
Octavio Filgueiras ◽  
Paul G. Casola ◽  
Don W. Nicholson ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Mitochondrial Activity ◽  
Rat Lung ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Mercuric Ions

CDPdiacylglycerol pyrophosphatase (E. C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6–8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 μM and Vmax values of 311 and 197 pmol∙min−1∙mg protein−1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

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