scholarly journals The determination and localization of sialic acid in guinea-pig granulocytes

1980 ◽  
Vol 192 (2) ◽  
pp. 543-550 ◽  
Author(s):  
J W DePierre ◽  
J Lazdins ◽  
M L Karnovsky
Keyword(s):  
Plasma Membrane ◽  
Sialic Acid ◽  
Guinea Pig ◽  
Total Cell ◽  
Polymorphonuclear Leucocytes ◽  
Intact Cells ◽  
Granule Fraction ◽  
Specific Determination ◽  
Chromatographic Procedure

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1–3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5–2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5′-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.

scholarly journals An Fc-receptor activity of plasma membranes from guinea-pig peritoneal polymorphonuclear leucocytes

1982 ◽  
Vol 204 (3) ◽  
pp. 625-634 ◽  
Author(s):  
S Tsunawaki ◽  
D Mizuno ◽  
K Kakinuma ◽  
M Kasahara
Keyword(s):  
Guinea Pig ◽  
Immune Complex ◽  
Plasma Membranes ◽  
Binding Activity ◽  
Fc Receptor ◽  
Receptor Activity ◽  
Alpha Amylase ◽  
Polymorphonuclear Leucocytes ◽  
Superoxide Generation ◽  
Intact Cells

Plasma membranes prepared from guinea-pig peritoneal polymorphonuclear leucocytes showed an immune-complex-binding activity that corresponded well with the activity in intact cells. The characteristics of this activity were reversible binding, dependence on the Fc portion of antigen-complexed IgG (immunoglobulin G), competition with aggregated IgG and independence from energy metabolism. These results support the conclusion that the binding activity found in the isolated plasma membranes is an Fc-receptor activity of guinea-pig peritoneal polymorphonuclear leucocytes. The activity showed Kd = 6.7×10(-8) M-IgG and maximum binding of 17 pmol of IgG/mg of membrane protein when measured with an immune complex of alpha-amylase and homologous guinea-pig anti-(alpha-amylase) IgG. Inhibition of the Fc-receptor activity by a series of various salts indicated the contribution of the hydrophobic interaction to the binding. Inhibitory effects of salts or metal-chelating reagents on the Fc-receptor activity were also observed on superoxide generation by these cells induced by the immune complex, suggesting a role of the Fc receptor as the immune-complex-binding site responsible for the initiation of superoxide generation.

scholarly journals The enzymic reduction and kinetics of oxidation of cytochrome b.245 of neutrophils

1982 ◽  
Vol 204 (2) ◽  
pp. 479-485 ◽  
Author(s):  
A R Cross ◽  
F K Higson ◽  
O T G Jones ◽  
A M Harper ◽  
A W Segal
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Room Temperature ◽  
Human Neutrophils ◽  
Polymorphonuclear Leucocytes ◽  
Myristate Acetate ◽  
Kinetics Of Oxidation ◽  
Granule Fraction ◽  
Kinetics Of ◽  
Stimulation Of

1. The absorption coefficient of human neutrophil plasma-membrane reduced-minus-oxidized cytochrome b-245 was determined [delta epsilon (mM; 559-540 nm) = 21.6 cm-1]. 2. Neutrophil polymorphonuclear leucocytes (neutrophils) were prepared from human, ox, horse and pig blood. In each case plasma-membrane fractions were found to contain low-potential cytochrome b. When membranes from horse neutrophils were incubated anaerobically with either NADH or NADPH the cytochrome b became reduced. Prior stimulation of the cells with phorbol myristate acetate did not increase the rate or extent of cytochrome b reduction in isolated membranes, but did increase both the rate and extent of reduction by NADPH in Triton-treated cells. 3. A cytochrome b was present also in the specific granule fraction of human neutrophils. Its Em (pH 7.0) was found to be -248 mV, very similar to that of the plasma-membrane cytochrome b. 4. The rate of oxidation of reduce cytochrome b-245 by air-saturated buffer, was determined by using stopped-flow techniques. In intact membranes t 1/2 for oxidation was 4.7 ms. This rate is sufficiently rapid to support the view that cytochrome b-245 is the oxidase in the respiratory burst of neutrophils. 5. Plasma-membrane cytochrome b of human neutrophils formed a complex with CO. At room temperature and 1 atm of CO approx. 40% of the cytochrome formed a complex; approx. 60% binding was measured at the increased concentration of dissolved CO achieved at 5 degrees C. The concentration of CO giving 50% binding was 1.18 mM.

scholarly journals The distributions of some granule-associated enzymes in guinea-pig polymorphonuclear leucocytes

1970 ◽  
Vol 116 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Robert H. Michell ◽  
Morris J. Karnovsky ◽  
Manfred L. Karnovsky
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Guinea Pig ◽  
Soluble Fraction ◽  
Maximum Activity ◽  
Polymorphonuclear Leucocyte ◽  
Urate Oxidase ◽  
Polymorphonuclear Leucocytes ◽  
Granule Fraction ◽  
Two Populations

1. Homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction. 2. The distributions in these fractions of protein, DNA, succinate dehydrogenase, β-glucuronidase, peroxidase, alkaline phosphatase, acid phosphatase (against p-nitrophenyl phosphate and β-glycerophosphate), cathepsin, and catalase were compared. 3. Almost all of the DNA sedimented in the first two pellets, indicating that the nuclei were relatively intact. 4. The four hydrolases and peroxidase showed different distribution patterns, although these activities were previously reported to be localized mainly in the single ‘granule’ fraction isolated from leucocytes. 5. The particles containing peroxidase, acid phosphatase and alkaline phosphatase all exhibited latency. Maximum activity for each enzyme was obtained at roughly similar concentrations of Triton X-100. 6. The acid phosphatase of these cells was distributed between two populations of particles that differed in both sedimentation characteristics and density. The acid phosphatase(s) of the two populations showed slightly different substrate specificities. This bimodal distribution was not an artifact of the procedure used to elicit the cells. 7. Catalase was recovered almost entirely in the soluble fraction and showed no latency in freshly prepared homogenates. No urate oxidase was detected. 8. We conclude that the ‘granule’ fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.

scholarly journals Different turnover of fucose residues in plasma membranes of rat liver and Morris hepatoma 7777

1980 ◽  
Vol 190 (1) ◽  
pp. 51-55 ◽  
Author(s):  
P Vischer ◽  
W Reutter
Keyword(s):  
Plasma Membrane ◽  
Sialic Acid ◽  
Rat Liver ◽  
Rate Constants ◽  
Plasma Membranes ◽  
Total Cell ◽  
Host Liver ◽  
Morris Hepatoma

Half-lives and rate constants of degradation of protein-bound fucose have been determined in plasma membranes and total cell homogenates of rat liver and Morris hepatoma 7777. The existence of at least two dynamically different classes of fucose-containing glycoproteins could be demonstrated in both liver and hepatoma plasma membranes. The apparent half-lives were 8.4 and 24.5 h (host liver) and 11.5 and 33.9 h (Morris hepatoma). Since this biphasic loss of fucose residues was not observed for sialic acid [Harms & Reutter (1974) Cancer Res. 34, 3165–3172], the differences are possibly related to specific functions of fucosylated glycoproteins of the plasma membrane.

COMPARISON OF VARIOUS METHODS FOR THE CHEMICAL ASSAY OF CORTICOIDS IN URINE

1955 ◽  
Vol 18 (4) ◽  
pp. 374-378
Author(s):  
Mogens Sprechler
Keyword(s):  
Calibration Curve ◽  
Periodic Acid ◽  
Reducing Power ◽  
Standard Technique ◽  
Phosphomolybdic Acid ◽  
Florisil Column ◽  
Chemical Assay ◽  
Chromatographic Procedure ◽  
Acid Reagent

SUMMARY Since 1949 about 10,000 urinary corticoid analyses have been performed routinely in our laboratory. The method used for this purpose was described in 1950 (Sprechler). We determine the corticoids which can be extracted from the urine with chloroform immediately after acidification to pH 1. The extract is washed with sodium hydroxide and water, a Girard separation is performed, and finally the reducing power of the ketonic fraction is measured by means of the phosphomolybdic acid reagent reaction. During the last few years two other chemical reactions have been used for comparison: The formaldehyde and the Porter-Silber method. After a thorough examination of the above methods a standard technique was followed. In the formaldehyde method a microdiffusion in a Conway unit was used instead of distillation of the formaldehyde following the oxidation with periodic acid. The calibration curve was corrected for loss of material by taking the standard doses of DOC through all the procedures of the method. A micromodification of the Porter-Silber method was chosen. Furthermore attempts were made to determine how specific the chromatographic procedure is in the determination of steroids in urinary extracts. For this purpose the Florisil column was used, and the technique described by Nelson & Samuels was followed. Finally we have investigated the glucuronide-bound corticoids in urine in a smaller series of objects.

COMPARATIVE ACTION OF VARIOUS OESTROGENIC COMPOUNDS ON MOUSE VAGINAL SIALIC ACIDS (II)

1969 ◽  
Vol 62 (4) ◽  
pp. 663-670 ◽  
Author(s):  
Lars Carlborg
Keyword(s):  
Sialic Acid ◽  
Response Curve ◽  
Sialic Acids ◽  
Clear Cut ◽  
Suitable Parameter ◽  
Sufficient Degree ◽  
Comparative Action ◽  
Relative Potencies ◽  
Good Agreement

ABSTRACT Oestrogens administered in lower doses than necessary to induce full cornification of the mouse vagina induce mucification. It was shown previously that the degree of mucification could be estimated by quantitative determination of sialic acids. A suitable parameter for oestrogen assay was the measurement of vaginal sialic acid concentration which exhibited a clear cut dose response curve. Eleven assays of various oestrogens were performed with this method. Their estimated relative potencies were in good agreement with other routine oestrogen assays. A statistically sufficient degree of precision was found. The sensitivity was of the same order, or slightly higher, than the Allen-Doisy test.

Improved Cleanup and Derivatization for Gas Chromatographic Determination of Monosodium Glutamate in Foods

1983 ◽  
Vol 66 (6) ◽  
pp. 1528-1531 ◽  
Author(s):  
Hiroshi Nakanishi
Keyword(s):  
Glutamic Acid ◽  
Acid Derivative ◽  
Monosodium Glutamate ◽  
Chromatographic Determination ◽  
Flame Ionization Detection ◽  
Gas Chromatograph ◽  
Flame Ionization ◽  
Acetone Water ◽  
Chromatographic Procedure

Abstract A gas chromatographic procedure is described for determining monosodium glutamate (MSG) in several types of food. A sample is extracted with acetone- water (1 + 1). Acetone is evaporated and an aliquot of the extract is buffered with 1M NH4OH-1M NH4CI pH 9 solution, and chromatographed directly on a column of QAE Sephadex A-25 that has been pretreated with the same buffer. MSG is eluted with 0.1N HC1, and a portion of the eluate is evaporated to dryness and reacted with dimethylformamide( DMF)-dimethylacetal to form the glutamic acid derivative, which is injected into a gas chromatograph and measured by flame ionization detection. Recoveries of MSG from sample fortified at 5-500 mg ranged from 92.8 to 100%.

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