STUDIES WITH STAPHYLOCOCCAL TOXINS: V. POSSIBLE IDENTIFICATION OF ALPHA HEMOLYSIN WITH A PROTEOLYTIC ENZYME

1960 ◽  
Vol 6 (2) ◽  
pp. 183-194 ◽  
Author(s):  
J. Robinson ◽  
F. S. Thatcher ◽  
Jeannine Montford
Keyword(s):  
Proteolytic Enzyme ◽  
Divalent Cations ◽  
Tetraacetic Acid ◽  
Sheep Erythrocytes ◽  
Alpha Hemolysin ◽  
Apparent Loss ◽  
Zone Electrophoresis ◽  
Chromatographic Procedure ◽  
Different Cultures ◽  
Hydrolyze Casein

Alpha hemolysin has been separated from many of the other components present in culture filtrates of four strains of Staphylococcus aureus using a column chromatographic procedure with carboxymethyl-cellulose as the selective adsorbent, and graded levels of phosphate buffer at pH 6.0 to provide selective elution. The preparation of alpha hemolysin obtained in this manner hemolyzed rabbit and sheep erythrocytes, induced a dermonecrotic reaction in rabbits, was lethal to mice, and was proteolytic. The hemolytic activity of the preparation was stimulated by ethylenediamine tetraacetic acid, presumably by removing toxic ions from solution. Divalent cations inhibited activity of the alpha hemolysin, but stimulated the activity of a distinctive sheep hemolysin, which is shown to be a separate entity.Alpha hemolysin, obtained by the chromatographic procedure, and subjected further to resolution by zone electrophoresis retained its capacity to induce dermonecrosis, to hemolyze rabbit erythrocytes, and to hydrolyze casein. Apparent loss of the capacity of the preparation to hemolyze sheep erythrocytes was discussed.The possibility that alpha hemolysin is a specific proteolytic enzyme is suggested by the observation that under no conditions could a separation be effected between the hemolytic and proteolytic properties of the alpha hemolysin recovered from a number of different cultures.

STUDIES WITH STAPHYLOCOCCAL TOXINS: VII. SEPARATION OF A PROTEOLYTIC ENZYME FROM ALPHA HEMOLYSIN

1963 ◽  
Vol 9 (5) ◽  
pp. 697-702 ◽  
Author(s):  
J. Robinson ◽  
F. S. Thatcher
Keyword(s):  
Ion Exchange ◽  
Ethyl Alcohol ◽  
Zinc Acetate ◽  
Proteolytic Enzyme ◽  
Potato Starch ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sheep Erythrocytes ◽  
Alpha Hemolysin ◽  
Zone Electrophoresis

Certain preparations of alpha hemolysin induced dissolution of rabbit and sheep erythrocytes and hydrolyzed casein. Components which were independently hemolytic and proteolytic were separated by the following sequence of procedures: precipitation with zinc acetate and ethyl alcohol at approximately −5 °C and pH 4.0, ion exchange chromatography with carboxymethylcellulose, and zone electrophoresis with granules of potato starch. The comparative lytic activity of the hemolytic component against erythrocytes of the rabbit and of the sheep was 19:1. No separation could be effected between these two hemolytic properties.

Divalent cations and vitamin B12 uptake by intestinal brush-border membrane vesicles

1980 ◽  
Vol 239 (6) ◽  
pp. G452-G456
Author(s):  
R. C. Beesley ◽  
C. D. Bacheller
Keyword(s):  
Vitamin B12 ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Divalent Cations ◽  
Intrinsic Factor ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Tetraacetic Acid ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border

Brush-border membrane vesicles from hamster intestine were employed to investigate uptake (binding) of vitamin B12 (B12). Ileal vesicles took up 25 times more B12 than did jejunal vesicles. Uptake of B12 by ileal vesicles was dependent on intrinsic factor (IF) and required Ca2+. Increasing the Ca2+ concentration caused an increase in uptake of B12 reaching a maximum at approximately 8 mM Ca2+. At high Ca2+ concentrations, 6–8 mM, Mg2+ had little effect on uptake of B12. At low Ca2+ concentrations, up to 2 mM, Mg2+ stimulated B12 uptake. Mg2+, Mn2+, and, to a lesser extent, Sr2+ stimulated Ca2+-dependent B12 uptake, but Zn2+, Ba2+, Na+, K+, and La3+ did not. B12 was apparently not metabolized and was bound as IF-B12 complex, which could be removed with (ethylenedinitrilo)tetraacetic acid (EDTA). Our results suggest that two types of divalent cation reactive sites are involved in binding of IF-B12. One is Ca2+ specific. The other is less specific reacting with Mg2+, Mn2+, Sr2+, and perhaps Ca2+ itself, thereby stimulating Ca2+-dependent binding of IF-B12 to its ileal receptor.

scholarly journals M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1111-1121 ◽  
Author(s):  
Beata Furmanek-Blaszk ◽  
Robert Boratynski ◽  
Natalia Zolcinska ◽  
Marian Sektas
Keyword(s):  
Functional Analysis ◽  
Divalent Cations ◽  
Restriction Enzymes ◽  
Bacterial Cells ◽  
Recognition Sequence ◽  
Moraxella Bovis ◽  
Chromatographic Procedure ◽  
Methylation Activity ◽  
Restriction Modification ◽  
Endonuclease Gene

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5′-GAAGA-3′/3′-CTTCT-5′. M1.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ to N 6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yielding N 4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000±1000 Da under denaturing conditions. Divalent cations (Ca2+, Mg2+, Mn2+ and Zn2+) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.

scholarly journals CRITIQUE ON THE K-PYROANTIMONATE METHOD FOR SEMIQUANTITATIVE ESTIMATION OF CATIONS IN CONJUNCTION WITH ELECTRON MICROSCOPY

1972 ◽  
Vol 20 (1) ◽  
pp. 65-78 ◽  
Author(s):  
RICHARD L. KLEIN ◽  
SHYUE-SHONG YEN ◽  
ÅSA THURESON-KLEIN
Keyword(s):  
Electron Microscopy ◽  
Divalent Cations ◽  
Test Tube ◽  
Tetraacetic Acid ◽  
Test Conditions ◽  
Solubility Products ◽  
Controlled Preparation ◽  
Preparation Techniques ◽  
Potassium Pyroantimonate

The histochemical method employing potassium pyroantimonate in conjunction with electron microscopy has been investigated using carefully controlled preparation techniques and very sensitive atomic absorption analysis of cations. A critique on the reliability and limitations of the method based on test tube and in vitro experiments is given. The method is sensitive to Ca++, Mg++ and Na+ at the 10–6, 10–5 and <10–2 M levels, respectively. Under defined conditions a linear ~l:l ratio of cation present to cation precipitated occurs above these levels. Approximate solubility products have been estimated. Under the test conditions, K+ does not precipitate as a pyroantimonate salt, and neither K+ nor OsO4 influences cation precipitaton at physiologic concentrations. Unbuffered, Tris-HCl-buffered and weakly buffered NaHCO3 media at pH 7.2-7.8 give statistically similar results with Na+ precipitation. The pyroantimonate ion can compete with chelators, ethylenedinitrilotetraacetic acid and ethylene glycol bis-N, N'-tetraacetic acid, for divalent cations when employed simultaneously. These chelators effectively remove Ca++ but not Mg++ from embryonic myocardium, and their effects on Na+ and K+ balance are not marked if employed for relatively short periods. Electron micrographic examples of cation precipitates are given in support of certain findings. A brief discussion of the significance of pyroantimonate grain size, the discrepancy between the ratio of intra- and extracellular precipitates and guidelines for the use of the method are included.

Reversible Changes of Chromosome Structure upon Different Concentrations of Divalent Cations

2019 ◽  
Vol 25 (3) ◽  
pp. 817-821 ◽  
Author(s):  
Astari Dwiranti ◽  
Hideaki Takata ◽  
Kiichi Fukui
Keyword(s):  
Electron Microscopy ◽  
Chromosome Structure ◽  
Ethylenediaminetetraacetic Acid ◽  
Divalent Cations ◽  
Chromosome Organization ◽  
Human Chromosomes ◽  
Tetraacetic Acid ◽  
Structural Details ◽  
Scanning Electron

AbstractThe structural details of chromosomes have been of interest to researchers for many years, but how the metaphase chromosome is constructed remains unsolved. Divalent cations have been suggested to be required for the organization of chromosomes. However, detailed information about the role of these cations in chromosome organization is still limited. In the current study, we investigated the effects of Ca2+ and Mg2+ depletion and the reversibility upon re-addition of one of the two ions. Human chromosomes were treated with different concentrations of Ca2+and Mg2+. Depletion of Ca2+ and both Ca2+ and Mg2+ were carried out using 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ethylenediaminetetraacetic acid (EDTA), respectively. Chromosome structure was examined by fluorescence microscopy and scanning electron microscopy. The results indicated that chromosome structures after treatment with a buffer without Mg2+, after Ca2+ depletion, as well as after depletion of both Mg2+, and Ca2+, yielded fewer compact structures with fibrous chromatin than those without cation depletion. Interestingly, the chromatin of EDTA-treated chromosomes reversed to their original granular diameters after re-addition of either Mg2+ or Ca2+ only. These findings signify the importance of divalent cations on the chromosome structure and suggest the interchangeable role of Ca2+ and Mg2+.

Passive K+-Cl- fluxes in low-K+ sheep erythrocytes: modulation by A23187 and bivalent cations

1985 ◽  
Vol 249 (3) ◽  
pp. C271-C278 ◽  
Author(s):  
P. K. Lauf
Keyword(s):  
Complete Inhibition ◽  
Cell Swelling ◽  
Ionophore A23187 ◽  
Tetraacetic Acid ◽  
Sheep Erythrocytes ◽  
Cl Transport ◽  
Bivalent Cations ◽  
Low K ◽  
O 10 ◽  
Stimulation Of

A fraction of the ouabain-resistant (OR) K+ flux of low-K+ (LK) sheep erythrocytes is Cl- dependent (K+-Cl- transport) and is activated reversibly by cell swelling or irreversibly by treatment with N-ethylmaleimide (NEM). The effect of the ionophore A23187 plus bivalent cations (Me2+) or ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) was studied on K+-Cl- transport in control or NEM-treated LK cells. The following observations were made. 1) A23187 (6 microM), at a hematocrit of 10% (vol/vol) and in the presence of 1 mM EGTA, activated severalfold OR K+-Cl- transport in shrunken or swollen cells but failed to stimulate further K+-Cl- flux in NEM-treated cells. 2) In the absence of EGTA, but at very low external Ca2+ concentrations [( Ca2+]o = 10(-7) M), A23187 stimulated OR K+-Cl- flux in controls less than with EGTA and inhibited it slightly in NEM-treated cells. 3) When [Ca2+]o was raised to 10(-3) M, an almost complete inhibition of OR K+-Cl- fluxes occurred in shrunken, swollen, or NEM-treated cells. 4) Other Me2+ inhibited OR K+-Cl- flux in the presence of A23187 in the following order of decreasing potency: Mn2+ much greater than Ca2+ greater than Mg2+ greater than Sr2+ much much greater than Ba2+. 5) Stimulation of OR K+-Cl- flux by A23187 +/- EGTA and inhibition by A23187 + Ca2+ were reversible and did not alter significantly cellular ATP. 6) The stimulatory effect of A23187 plus EGTA, perhaps by Me2+ removal, on K+-Cl- flux and its inhibition by Ca2+ were reversibly abolished in metabolically depleted cells.(ABSTRACT TRUNCATED AT 250 WORDS)

STUDIES WITH STAPHYLOCOCCAL TOXINS: VI. SOME PROPERTIES OF A NON-HEMOLYTIC DERMONECROTIC TOXIN PRODUCED BY SOME STRAINS OF STAPHYLOCOCCUS AUREUS

1960 ◽  
Vol 6 (2) ◽  
pp. 195-201 ◽  
Author(s):  
J. Robinson ◽  
F. S. Thatcher ◽  
Jeannine Montford
Keyword(s):  
Staphylococcus Aureus ◽  
Proteolytic Activity ◽  
Carboxymethyl Cellulose ◽  
Dermonecrotic Toxin ◽  
Culture Filtrates ◽  
Zone Electrophoresis ◽  
Chromatographic Procedure ◽  
Selective Adsorbent

A non-hemolytic, dermonecrotic preparation with proteolytic activity was isolated from culture filtrates of some strains of Staphylococcus aureus using a chromatographic procedure with carboxymethyl-cellulose as a selective adsorbent. The preparation was resolved into components which were independently proteolytic and dermonecrotic using a zone electrophoresis procedure.The non-hemolytic dermonecrotic toxin possessed properties which characterize it as a protein. No relationship could be found between pathogenicity of the staphylococcus and its capacity to produce the toxin.The presence in some culture filtrates of the non-hemolytic, dermonecrotic toxin has been considered as a possible explanation for the lack of agreement noted by several authors between alpha hemolytic and dermonecrotic titers.

Activation and inhibition of the brush-border membrane-bound alkaline phosphatase activity of Hymenolepis diminuta (Cestoda) by divalent cations

Parasitology ◽  
1991 ◽  
Vol 102 (1) ◽  
pp. 141-145 ◽  
Author(s):  
P. W. Pappas
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Brush Border ◽  
Divalent Cations ◽  
Hymenolepis Diminuta ◽  
Tetraacetic Acid ◽  
Low Concentrations ◽  
Membrane Bound ◽  
High Concentrations

SUMMARYIn the absence of exogenous divalent cations, the isolated brush-border plasma membrane of Hymenolepis diminuta possesses alkaline phosphatase activity (APA). APA is stimulated in the presence of exogenous Mg2+ and inhibited by low concentrations of Zn2+ or high concentrations of Ca2+, and inhibition of APA by Zn2+ is reversed by both Mg2+ and Ca2+. APA is inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid, and 1, 10-phenanthroline in time- and concentration-dependent fashions, with EDTA being the most effective inhibitor. Following treatment with EDTA, APA is restored by Mg2+ and, to a lesser extent, by Ca2+, but not by Zn2+. Thus, APA represents a Mg2+-dependent enzyme that can be partly activated by Ca2+ but only in the absence of Mg2+.

Permeation of divalent cations through the Ca2+ channel of rabbit portal vein myocytes

1992 ◽  
Vol 262 (2) ◽  
pp. H326-H330
Author(s):  
D. A. Katzka ◽  
R. Cox ◽  
A. J. Davidoff ◽  
M. Morad
Keyword(s):  
Portal Vein ◽  
Ethylene Glycol ◽  
Divalent Cations ◽  
Concentration Addition ◽  
Ion Permeation ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Maximum Current ◽  
Rabbit Portal Vein

The divalent selectivity of the Ca2+ channel in the rabbit portal vein myocyte was examined by the whole cell clamp method. A concentration-dependent selectivity of divalent ion permeation was found such that when Ca2+ was replaced by Ba2+ or Sr2+, the order of maximum current was Ca2+ = Ba2+ greater than Sr2+ at 2 mM and Ba2+ greater than Sr2+ greater than or equal to Ca2+ at 5-10 mM. The possibility of block of the Ca2+ channel by micromolar concentrations of "contaminant" Ca2+ as a determinant of change in the order of selectivity of divalents was examined. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (500 microM) significantly increased maximum Ba2+ current (IBa) or ISr in solution containing 5 mM Ba2+ or Sr2+. Furthermore, at 5 mM extracellular Ba2+ concentration, addition of 10, 20, 50, and 100 microM Ca2+ caused a 6, 14, 22, and 33% decrease in IBa, respectively. These results suggest that the portal vein Ca2+ channel has three orders of magnitude higher selectivity for Ca2+ over Ba2+ and Sr2+ such that micromolar Ca2+ may block permeation of other divalents through the channel.

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