Preliminary Characterization of Phospholipase A 2 in Lagenidium giganteum

Joanna K. MacKichan, Amy Tuininga, James L. Kerwin

Research output: Contribution to journalArticleResearchpeer-review

3 Citations (Scopus)

Abstract

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A 2 in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A 2 (PLA 2 ) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA 2 activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA 2 activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1- 14 C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca 2+ , Mg 2+ , and Mn 2+ all enhanced PLA 2 activity, while Co 2+ , Fe 2+ , and Zn 2+ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca 2+ and Mn 2+ than Mg 2+ , reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA 2 activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA 2 hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA 2 activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA 2 activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.

Original languageEnglish
Pages (from-to)180-192
Number of pages13
JournalExperimental Mycology
Volume18
Issue number2
DOIs
StatePublished - 1 Jun 1994

Fingerprint

Lagenidium
Phospholipases A
Egtazic Acid
Enzymes
Chelating Agents
Edetic Acid
Hydrolysis
Mycology
Ellagic Acid
Masoprocol
Gossypol
Glycerophospholipids
Cell Separation
Mycelium
Divalent Cations
Sterols
Life Cycle Stages
Phosphatidylcholines
Chromatography
Phospholipids

Keywords

  • Lagenidium giganteum
  • Lipase
  • Oomycetes
  • cholesterol
  • oosporogenesis
  • phosphatidylcholine
  • phospholipase A
  • phospholipids

Cite this

MacKichan, Joanna K. ; Tuininga, Amy ; Kerwin, James L. / Preliminary Characterization of Phospholipase A 2 in Lagenidium giganteum In: Experimental Mycology. 1994 ; Vol. 18, No. 2. pp. 180-192.
@article{7a0edd6a6e60451da3c402a23fddd001,
title = "Preliminary Characterization of Phospholipase A 2 in Lagenidium giganteum",
abstract = "MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A 2 in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A 2 (PLA 2 ) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA 2 activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA 2 activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1- 14 C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca 2+ , Mg 2+ , and Mn 2+ all enhanced PLA 2 activity, while Co 2+ , Fe 2+ , and Zn 2+ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca 2+ and Mn 2+ than Mg 2+ , reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA 2 activity by about 80{\%} at 5 mM concentration, 50{\%} with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA 2 hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA 2 activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA 2 activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.",
keywords = "Lagenidium giganteum, Lipase, Oomycetes, cholesterol, oosporogenesis, phosphatidylcholine, phospholipase A, phospholipids",
author = "MacKichan, {Joanna K.} and Amy Tuininga and Kerwin, {James L.}",
year = "1994",
month = "6",
day = "1",
doi = "10.1006/emyc.1994.1019",
language = "English",
volume = "18",
pages = "180--192",
journal = "Experimental Mycology",
issn = "0147-5975",
publisher = "Academic Press Inc.",
number = "2",

}

Preliminary Characterization of Phospholipase A 2 in Lagenidium giganteum . / MacKichan, Joanna K.; Tuininga, Amy; Kerwin, James L.

In: Experimental Mycology, Vol. 18, No. 2, 01.06.1994, p. 180-192.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Preliminary Characterization of Phospholipase A 2 in Lagenidium giganteum

AU - MacKichan, Joanna K.

AU - Tuininga, Amy

AU - Kerwin, James L.

PY - 1994/6/1

Y1 - 1994/6/1

N2 - MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A 2 in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A 2 (PLA 2 ) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA 2 activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA 2 activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1- 14 C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca 2+ , Mg 2+ , and Mn 2+ all enhanced PLA 2 activity, while Co 2+ , Fe 2+ , and Zn 2+ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca 2+ and Mn 2+ than Mg 2+ , reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA 2 activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA 2 hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA 2 activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA 2 activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.

AB - MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A 2 in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A 2 (PLA 2 ) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA 2 activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA 2 activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1- 14 C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca 2+ , Mg 2+ , and Mn 2+ all enhanced PLA 2 activity, while Co 2+ , Fe 2+ , and Zn 2+ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca 2+ and Mn 2+ than Mg 2+ , reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA 2 activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA 2 hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA 2 activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA 2 activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.

KW - Lagenidium giganteum

KW - Lipase

KW - Oomycetes

KW - cholesterol

KW - oosporogenesis

KW - phosphatidylcholine

KW - phospholipase A

KW - phospholipids

UR - http://www.scopus.com/inward/record.url?scp=0028225259&partnerID=8YFLogxK

U2 - 10.1006/emyc.1994.1019

DO - 10.1006/emyc.1994.1019

M3 - Article

VL - 18

SP - 180

EP - 192

JO - Experimental Mycology

JF - Experimental Mycology

SN - 0147-5975

IS - 2

ER -