scholarly journals Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase

1980 ◽  
Vol 187 (1) ◽  
pp. 197-204 ◽  
Author(s):  
W A Gahl ◽  
A M Vale ◽  
H C Pitot
Keyword(s):  
Bovine Serum ◽  
Oxidase Activity ◽  
Gel Filtration ◽  
Major Product ◽  
Sephadex Column ◽  
Exchange Column ◽  
Cation Exchange Column ◽  
Putrescine Oxidase ◽  
Spermidine Oxidase ◽  
Foetal Bovine Serum

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.

scholarly journals Polyamine degradation in foetal and adult bovine serum

1982 ◽  
Vol 202 (3) ◽  
pp. 603-611 ◽  
Author(s):  
W A Gahl ◽  
H C Pitot
Keyword(s):  
Bovine Serum ◽  
Oxidase Activity ◽  
Degrading Enzyme ◽  
Substrate Preferences ◽  
Adult Bovine Serum ◽  
Putrescine Oxidase ◽  
20 Nm ◽  
Spermidine Oxidase ◽  
Foetal Bovine Serum

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone) (MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.

scholarly journals Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase

1982 ◽  
Vol 201 (1) ◽  
pp. 161-166 ◽  
Author(s):  
W A Gahl ◽  
A M Vale ◽  
H C Pitot
Keyword(s):  
Oxidase Activity ◽  
Diamine Oxidase ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Human Pregnancy ◽  
Diamine Oxidase Activity ◽  
Radioactive Assay ◽  
Spermidine Oxidase ◽  
In Pregnancy

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.

scholarly journals Foetal bovine serum influence on in vitro extracellular vesicle analyses

2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Brandon M. Lehrich ◽  
Yaxuan Liang ◽  
Massimo S. Fiandaca
Keyword(s):  
Bovine Serum ◽  
Extracellular Vesicle ◽  
Foetal Bovine Serum

scholarly journals Structural similarity of chymopapain forms as indicated by circular dichroism

1989 ◽  
Vol 257 (1) ◽  
pp. 183-186 ◽  
Author(s):  
S Solis-Mendiola ◽  
R Zubillaga-Luna ◽  
A Rojo-Dominguez ◽  
A Hernandez-Arana
Keyword(s):  
Liquid Chromatography ◽  
High Resolution ◽  
Cation Exchange ◽  
Proteolytic Activity ◽  
Structural Similarity ◽  
Tertiary Structures ◽  
Exchange Column ◽  
Cation Exchange Column ◽  
Modified Proteins ◽  
Circular Dichroïsm

Four chymopapain forms were isolated by high-resolution liquid chromatography on a cation-exchange column. The three major forms possess nearly identical secondary and tertiary structures, as judged from their c.d. spectra; these components showed similar proteolytic activity and Mr values close to that of papain. The fourth isolated component seems to be a mixture of modified proteins.

scholarly journals Clearance and binding of native and defucosylated lactoferrin

1983 ◽  
Vol 212 (2) ◽  
pp. 249-257 ◽  
Author(s):  
M J Imber ◽  
S V Pizzo
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Specific Binding ◽  
Gel Filtration ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocyte ◽  
Stable Complex

These studies explore the role of carbohydrate recognition systems and the direct involvement of terminal alpha 1-3-linked fucose in the clearance of lactoferrin from the murine circulation and in the specific binding of lactoferrin to receptors on murine peritoneal macrophages. As previously reported, radiolabelled lactoferrin cleared very rapidly (t1/2 less than 1 min) after intravenous injection into mice. However, competing levels of ligands specific for the hepatic galactose receptor (asialo-orosomucoid), the hepatic fucose receptor (fucosyl-bovine serum albumin), and the mononuclear-phagocyte system pathway recognizing mannose, N-acetylglucosamine and fucose (mannosyl-, N-acetylglucosaminyl- and fucosyl-bovine serum albumin) did not block radiolabelled lactoferrin clearance in vivo or binding to mouse peritoneal macrophage monolayers in vitro. Almond emulsin alpha 1-3-fucosidase was used to prepare defucosylated lactoferrin in which 88% of the alpha 1-3-linked fucose was hydrolysed. No difference in clearance or receptor binding was observed between radiolabelled native and defucosylated lactoferrin. Fucoidin, a fucose-rich algal polysaccharide, completely inhibits the clearance in vivo and macrophage binding in vitro of lactoferrin. This effect, however, is probably not the result of competition for binding to the fucose receptor, since gel-filtration studies demonstrated formation of a stable complex between lactoferrin and fucoidin. The present results indicate that the lactoferrin-clearance pathway is distinct from several pathways mediating glycoprotein clearance through recognition of terminal galactose, fucose, N-acetylglucosamine or mannose. Furthermore, alpha 1-3-linked fucose on lactoferrin is not essential for lactoferrin clearance in vivo or specific binding to macrophage receptors in vitro.

Hemoglobin A1C by Hplc with the Pharmacia Mono S HR 5/N Cation-Exchange Column: Influence of Sample Protein Load on Optimal Chromatographic Conditions

1992 ◽  
Vol 38 (8) ◽  
pp. 1488-1490 ◽  
Author(s):  
J C Philcox ◽  
M R Haywood ◽  
A M Rofe
Keyword(s):  
Cation Exchange ◽  
Flow Rate ◽  
Chromatographic Separation ◽  
Hemoglobin A1c ◽  
Analysis Time ◽  
Exchange Column ◽  
Cation Exchange Column ◽  
Protein Load

Abstract The Pharmacia Mono S HR 5/5 column has been optimized for hemoglobin A1c analysis by HPLC by using a much smaller column load, decreased buffer flow rate, and a steeper gradient than was used in previously described methods. Superior chromatographic separation, shorter analysis time, and a greatly extended column life have resulted.

scholarly journals Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss)

2019 ◽  
Vol 64 (No. 12) ◽  
pp. 547-557
Author(s):  
H Minarova ◽  
M Palikova ◽  
J Mares ◽  
E Syrova ◽  
J Blahova ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Bovine Serum ◽  
Lymphocyte Proliferation ◽  
Head Kidney ◽  
Pokeweed Mitogen ◽  
Lymphocyte Proliferation Assay ◽  
Proliferation Assay ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Foetal Bovine Serum

The lymphocyte proliferation assay is a valuable method used for the evaluation of the fish immune system. However, there are many variations and optimal results are not always obtained. Unification is necessary to ensure the comparability between different studies. The aim of this study was to optimise the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). This goal included the determination of the optimal incubation length, serum type, incubation temperature, type of mitogen and its concentration, and anticoagulant. The peripheral blood and head kidney lymphocytes were isolated by density gradient centrifugation. Subsequently, the cells were incubated for 3–8 days with different mitogens (pokeweed mitogen 5, 10 and 50 µg/ml, concanavalin A 1, 10 and 20 µg/ml, phytohaemagglutinin 25, 50 and 100 µg/ml, lipopolysaccharide 1, 50 and 100 µg/ml). The use of the different serum types (foetal bovine serum, trout serum), incubation temperatures (10–20 °C) and anticoagulants (heparin, EDTA) was compared. Labelled thymidine was used to evaluate the assay. The best results were obtained after seven days of incubation at 15 °C with foetal bovine serum (FBS). The head kidney lymphocytes showed the highest proliferative response with 50 µg/ml phytohaemagglutinin. With the peripheral blood lymphocytes (heparin and EDTA), the best results were obtained with 50 µg/ml pokeweed mitogen. The highest proliferation levels were detected with heparinised blood. In conclusion, optimisation of this assay contributes to the improved assessment of the rainbow trout immune function.

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