Biochemical SocietyTransactions ( 1 992)20
A study of the 14kDa Phospholipase A, from rat liver using a continuous fluorescent displacement assay for the enzyme
250
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w.
c
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ADRIAN R. KINKAID and DAVID C. WILTON
287s
ad 0
U
Department of Biochemistry, University of Southampton, SO9 3TU. UK
I
- 04 c
100
- 0.4
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-.-n p
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150
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Low molecular weight phospholipase A, is a ubiquitous protein with an absolute requirement for calcium. It hydrolyses the sn-2 ester bond of a wide range of phospholipids and is found at raised concentrations in inflammatory diseases such as arthritis. Rat liver contains relatively high levels of the enzyme, where it is bound predominantly to the mitochondria1 fraction after homogenisation of whole liver. This membrane-bound PLA, can be released by sonication in the presence of high salt (1M KCI). Once solubilised the enzyme activity can be measured using a fluorescence displacement assay [I]. In this communication we describe the partial purification of the enzyme following essentially the method of Aarsman et al. [2] for the initial steps and using the fluorescence assay to monitor phospholipase A, activity during the chromatographic procedures. Further purification was achieved by heparin-Sepharose chromatography previously used with the rat platelet enzyme [3]. Studies on the substrate specificity of the enzyme (see below) have identified the effectiveness of dioleoyl-phosphatidyl glycerol as a substrate for enzyme assays. The mitochondrial fraction was prepared from male Wistar albino rat liver and the phospholipase A, activity released by The released activity was treatment with 1M KCI [2]. fractionated on a Sephadex (3-75 column (26 x 900 mm), equilibrated with release buffer (1 M KCI, 0.25 M Sucrose, 100 mM Tris-HCI, pH 7.4, I mM EDTA). Eluted fractions were collected and assayed for phospholipase activity. The elution profile from the Sephadex G-75 column revealled the bulk of activity to be in the low molecular weight region of the eluate. Active fractions were pooled and concentrated using an Amicon YM5 ultrafiltration membrane. The concentrate was then diluted 5 times to reduce the ionic strength and concentrated further to about 25 ml. The concentrate was loaded onto a heparin-Sepharose column (16 x 90 mm) and eluted with a KCI gradient gradient. Fractions were collected and assayed for enzyme activity and the elution profile is shown in Fig. 1. Active fractions were pooled and concentrated by ultrafiltration with an Amicon YM5 membrane. This partially purified preparation of phospholipase A, was used to determine the substrate specificity of the enzyme using several different naturally ocurring phospholipid substrates according to procedures described previously for other phospholipases A, [4].A comparison of rates of hydrolysis of dioleoyl-phosphatidyl glycerol, dioleoyl-phosphatidyl choline and dioleoyl-phosphatidyl ethanolamine using the fluorescent displacement assay is shown in Table 1.
Enzyme Rat Liver PLA,
Rate for DOPG
Rate for DOPE
Rate for DOPC
0.354
0.117
A study of the 14kDa Phospholipase A, from rat liver using a continuous fluorescent displacement assay for the enzyme
250
y A1.0
'5 200
- 0.6
w.
c
.-
ADRIAN R. KINKAID and DAVID C. WILTON
287s
ad 0
U
Department of Biochemistry, University of Southampton, SO9 3TU. UK
I
- 04 c
100
- 0.4
a
-.-n p
-5-
150
n
Low molecular weight phospholipase A, is a ubiquitous protein with an absolute requirement for calcium. It hydrolyses the sn-2 ester bond of a wide range of phospholipids and is found at raised concentrations in inflammatory diseases such as arthritis. Rat liver contains relatively high levels of the enzyme, where it is bound predominantly to the mitochondria1 fraction after homogenisation of whole liver. This membrane-bound PLA, can be released by sonication in the presence of high salt (1M KCI). Once solubilised the enzyme activity can be measured using a fluorescence displacement assay [I]. In this communication we describe the partial purification of the enzyme following essentially the method of Aarsman et al. [2] for the initial steps and using the fluorescence assay to monitor phospholipase A, activity during the chromatographic procedures. Further purification was achieved by heparin-Sepharose chromatography previously used with the rat platelet enzyme [3]. Studies on the substrate specificity of the enzyme (see below) have identified the effectiveness of dioleoyl-phosphatidyl glycerol as a substrate for enzyme assays. The mitochondrial fraction was prepared from male Wistar albino rat liver and the phospholipase A, activity released by The released activity was treatment with 1M KCI [2]. fractionated on a Sephadex (3-75 column (26 x 900 mm), equilibrated with release buffer (1 M KCI, 0.25 M Sucrose, 100 mM Tris-HCI, pH 7.4, I mM EDTA). Eluted fractions were collected and assayed for phospholipase activity. The elution profile from the Sephadex G-75 column revealled the bulk of activity to be in the low molecular weight region of the eluate. Active fractions were pooled and concentrated using an Amicon YM5 ultrafiltration membrane. The concentrate was then diluted 5 times to reduce the ionic strength and concentrated further to about 25 ml. The concentrate was loaded onto a heparin-Sepharose column (16 x 90 mm) and eluted with a KCI gradient gradient. Fractions were collected and assayed for enzyme activity and the elution profile is shown in Fig. 1. Active fractions were pooled and concentrated by ultrafiltration with an Amicon YM5 membrane. This partially purified preparation of phospholipase A, was used to determine the substrate specificity of the enzyme using several different naturally ocurring phospholipid substrates according to procedures described previously for other phospholipases A, [4].A comparison of rates of hydrolysis of dioleoyl-phosphatidyl glycerol, dioleoyl-phosphatidyl choline and dioleoyl-phosphatidyl ethanolamine using the fluorescent displacement assay is shown in Table 1.
Enzyme Rat Liver PLA,
Rate for DOPG
Rate for DOPE
Rate for DOPC
0.354
0.117
