Purification of human placental alkaline phosphatase by isoelectric focusing

Usategui-Gomez, M.; Yeager, Frances M.; Tarbutton, P. N.

doi:10.1016/0009-8981(74)90160-0

Cited by 15 publications

(3 citation statements)

References 13 publications

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“…A 1000-fold increase in enzyme specific activity in extracts from both sham-operated and cholestasisinduced enzyme was used to show the induced enzyme is regulated in a manner similar to the cortisolinduced HeLa enzyme by altering the enzyme intrinsic activity [37]. Human placental alkaline phosphatase was purified by isoelectric focusing and was judged to be homogeneous based on the following criteria [38] : (a) a 10-fold increase in specific activity, (b) a single band of protein and enzyme activity on polyacrylamide disc gel electrophoresis and (c) migration as a single band in sedimentation velocity gradients with no extraneous peaks or asymmetries indicative of contaminating proteins. Isoelectric focusing also inactivated the placental enzyme and activity could be partially restored by neutralizing the acidic fractions as soon as they were collected and by incubating them in the cold with Zn2+ [38].…”

Section: Discussionmentioning

confidence: 99%

“…Human placental alkaline phosphatase was purified by isoelectric focusing and was judged to be homogeneous based on the following criteria [38] : (a) a 10-fold increase in specific activity, (b) a single band of protein and enzyme activity on polyacrylamide disc gel electrophoresis and (c) migration as a single band in sedimentation velocity gradients with no extraneous peaks or asymmetries indicative of contaminating proteins. Isoelectric focusing also inactivated the placental enzyme and activity could be partially restored by neutralizing the acidic fractions as soon as they were collected and by incubating them in the cold with Zn2+ [38]. Reactivation of HeLa alkaline phosphatase following isoelectric focusing was not attempted in our study since the enzyme preparations were used for chemical analysis.…”

Section: Discussionmentioning

confidence: 99%

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Cortisol Modification of HeLa 65 Alkaline Phosphatase

Bazzell

Price

et al. 1976

European Journal of Biochemistry

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Alkaline phosphatase activity of HeLa cells is increased 5–20‐fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one‐half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Cortisol Modification of HeLa 65 Alkaline Phosphatase

Bazzell

Price

et al. 1976

European Journal of Biochemistry

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“…Es zeigte sich jedoch im Trennergebnis sowohl hinsichtlich der Zahl als auch der Lokalisation der Glutathion-Reduktase-Banden kein Unterschied im Vergleich zum Serum ohne FAD-Vorinkubation. Somit ist die durch Gel-Elektrophorese nachweisbare Heterogenität der Glutathion-Reduktase im Serum nicht auf einen unterschiedlichen Sättigungsgrad des Enzyms mit FAD zurückzuführen.Eine Änderung der Wanderungsgeschwindigkeit nach Inkubation mit Neuraminidase (23), wie sie für einige Isoenzyme im Serum beschrieben wurde(24), war bei keiner der 3 Glutathion-Reduktäse-Fraktionen zu beobachten. Damit kann die glykosidische Bindung der Glutathion-Reduktase an Neuraminsäure als mög-liche Ursache für die Ausbildung multipler Formen des Enzyms ausgeschlossen werden.Untersucht man Seren mit erhöhter Glutäthicm-Reduktase-Aktivität mittels Gelfiltration, so erhält man wie bei der Gelelektrophorese bis zu 3 Glutathion-ReduktaseFraktionen.…”

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Multiple Formen der NADPH-abhängigen Glutathion-Reduktase im Serum. Untersuchungen über die NADPH-abhängige Glutathion-Reduktase im Serum, II. Mitteilung

Weidemann¹,

Anselstetter²

1977

Clinical Chemistry and Laboratory Medicine

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Zusammenfassung: Seren mit erhöhter Glutathion-Reduktase-Aktivität wurden gelelektrophoretisch in Agar bzw. Polyacrylamid sowie mittels Gelfiltration untersucht. Beide Trennverfahren ergaben, daß sich die Glutathion-Reduktase-Aktivität in den einzelnen Proben auf bis zu 3 nach Wanderungsgeschwindigkeit und Molekülgröße unterschiedliche Fraktionen verteilt-Dabei entsprechen die Fraktionen mit dem kleinsten bzw. größten Molekulargewicht den am langsamsten bzw. am schnellsten wandernden Banden in der Agarelektrophorese. Vorinkubation der Serumproben mit FAD bzW. Neuraminidase hat keinen Einfluß auf die Wanderungsgeschwindigkeiten der 3 Fraktionen. Nach Zusatz von j3-Mercaptoethanol zum Serum findet man bei der Gelelektrophorese und der Gelfiltration nur noch die GlutathionReduktase-Fraktion mit der langsamsten Wanderungsgeschwindigkeit und dem niedrigsten Molekulargewicht (140 000). Nach Entfernung des Thiols werden die beiden anderen Glutathion-Reduktase-Fraktionen wieder nachweisbar. Weitere UiltefBuchungen an den nach Agargelelektrophorese isolierten Glutathion-Reduktase-Fraktionen zeigen, daß es sich bei den 3 Fraktionen der Glutathion-Reduktase im Serum um oligomere Formen des Enzyms handelt, die reversibel ineinander übergehen bzw. überführt werden können. Multiple forms of NADPH-dependent glutäthione reductase in serum. Studies on the NADPH-dependent glutathionereductase in serum II.Summary: Sera with elevated activities of glutäthione reductase were investigated by gel electrophoresis in agar or polyacrylamide, and by gel filtration. In both separation methods the glutäthione reductase activity in individual samples was resolved, showing up to three fractions differing in rate of migration and molecular size. The fractions with the lowest and highest molecular weight, corresponded respectively to the slowest and fastest migrating bands in agar gel electrophoresis. Preincubation of the serum samples with FAD or neuraminidase had no effect on the rate of migration of the three fractions. After the addition of ß-mercaptoethanol to the serum, gel electrophoresis and gel filtration showed only the enzyme fraction with the slowest rate of migration and the lowest molecular weight (140jOOO), The other two fractions reappeared after removal of the thiol from the serum. Further studies on the isolated (agar gel electrophoresis) fractions showed the existence of oligomeric forms of the enzyme, which are reversibly interconvertible. Einführung

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The value of a sensitive assay of carcino‐placental alkaline phosphatase (CPAP) in the follow‐up of gynecological cancers

Haije¹,

Meerwaldt²,

Talerman³

et al. 1979

Intl Journal of Cancer

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Using a sensitive enzyme immunoassay, carcinoplacental alkaline phosphatase (CPAP) was determined in sera of 1266 patients with gyneocological cancers. All these patients were referred after initial surgical treatment elsewhere. There were 95 patients with evidence of disease at the time of the study and 1171 without evidence of disease. Of the 95 patients with active disease, 47 were treated for ovarian carcinoma, 36 for carcinoma of the cervix and 12 for endometrial carcinoma. Raised levels of CPAP were seen in 40% of patients with ovarian carcinoma, in 22% with carcinoma of the cervix and in 41% in the small group with endometrial carcinoma. In patients without evidence of disease, raised levels of CPAP were seen in 12% of patients with carcinoma of the cervix, in 6% of endometrial carcinoma and only in 2% of patients with carcinoma of the ovary. Therefore it was considered that in the latter group CPAP studies would prove of some value. In the group of patients with carcinoma of the ovary and evidence of disease, raised levels of CPAP were seen almost exclusively in patients with epithelial tumors. It is considered that CPAP may be of value as a tumor marker in this group of patients. When compared with CEA, CPAP tends to give fewer false positives and correlates better with the presence of disease.

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Purification of human placental alkaline phosphatase by isoelectric focusing

Cited by 15 publications

References 13 publications

Cortisol Modification of HeLa 65 Alkaline Phosphatase

Cortisol Modification of HeLa 65 Alkaline Phosphatase

Multiple Formen der NADPH-abhängigen Glutathion-Reduktase im Serum. Untersuchungen über die NADPH-abhängige Glutathion-Reduktase im Serum, II. Mitteilung

The value of a sensitive assay of carcino‐placental alkaline phosphatase (CPAP) in the follow‐up of gynecological cancers

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