Measurements were made of the specific activities of the following glycerolipid synthesising enzymes; fatty acyl-CoA synthetase (FAS), microsomal and mitochondrial forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT), phosphatidate phosphohydrolase (PAP) 1 and 2, and diacylglycerol acyltransferase (DGAT). These were made in homogenates of ventricle muscle and of isolated ventricular myocytes in order to compare myocyte activities with those in the whole ventricle. This comparison has not previously been made and is relevant since many of these enzymes have high specific activities in adipocytes which are present in whole heart preparations. The assay methods established for use with white adipose tissue (WAT) and liver were adapted for the assessment of cardiac enzyme activities as described in section 2.11.
In the case of GPAT and PAP, further studies were undertaken in order to investigate some of the properties and subcellular distribution of these enzymes in heart, and to make comparisons with those more extensively characterised in liver and WAT.
3.1.1 Glycerolphosphate Acyltransferase
As previously discussed, two distinct forms of GPAT occur in liver and WAT, one localised in mitochondrial membranes and another in the endoplasmic reticulum. The mitochondrial enzyme is highly specific for saturated acyl-CoA and is resistant to thiol group reagents such as N-ethylmalemide (NEM). In contrast, microsomal GPAT will use both saturated and unsaturated acyl-CoA and is completely inhibited by NEM. These properties permit the two forms to be discriminated in crude tissue extracts.
Briefly, a high concentration (120 |aM) o f o leoy l-C oA measures only the microsomal enzym e, whereas a low er concentration (40 |iM ) o f palm itoyl-C oA in the presence o f NEM measures only the mitochondrial GPAT. Although the presence o f N E M -sensitive (presumably m icrosomal) and N E M -insensitive (presumably mitochondrial) forms o f G PAT have been reported to occur in rat heart (Heathers et al, 1985), it w as not known whether the substrate specificities observed in liver and W AT (or the assays described above) also applied to these enzym es in cardiac muscle.
Table 3 .1a sh ow s the properties o f subcellular fractions isolated from ventricle muscle. The sarcoplasmic reticulum (SR ) fraction w as enriched in SR (Ca^ /K -A T Pase) and microsomal (N A D P -cytochrom e c reductase) marker enzym e activities compared to the w h ole hom ogenate. Enrichment o f the (inner) mitochondrial membrane marker enzyme, succinate-cytochrom e c reductase, w as observed in the mitochondrial fraction.
A significant amount o f GPAT activity w as present in both o f these fractions. GPAT in the SR fraction was equally active with saturated and unsaturated acyl-C oA , and was inhibited over 95% by inclusion o f N E M in the assay buffer (table 3.2). In contrast, N E M -insensitive GPAT activity in the mitochondrial fraction w as highly selective for the saturated palm itoyl-C oA substrate. This indicates that the previously optimised assay conditions are suitable for use in heart extracts. The SR fraction w as enriched in the G PAT active with oleoyl-C oA compared with the N E M -insensitive, palm itoyl-CoA requiring activity (table 3.1b). The mitochondrial fraction exhibited an opposite enrichment profile.
Therefore, it can be concluded that heart SR contains a G PAT activity similar to the liver and W AT microsomal enzym e, whilst the GPAT in heart mitochondrial membranes has similar properties to the mitochondrial enzym e in liver and W AT.
Table 3.1a Distribution of marker enzymes in heart subcellular fractions
Subcellular fractions were obtained from homogenates of ventricle muscle. The values are expressed as nmol/min/mg of protein and are means of 2-3 independent preparations.
Ca^^/K^- Succinate-cyt c Ratio 1:2 NADlPycyt c ATPase (1) reductase (2) reductase
Whole homogenate 43 Mitochondria 35 Sarcoplasmic reticulum 194 29 93 50 1.5 0.5 3.9 4.5 6.6 40.3
Table 3.1b Subcellular distribution of GPAT in heart
Subcellular fractions were obtained from homogenates of ventricle muscle. Assays contained 40 pM palmitoyl Co A and 10 mM NEM or 120 pM oleoyl-CoA. The values are expressed as nmol/min/mg of protein and are means of 2 independent preparations.
With oleoyl-CoA (1) With palmitoyl-CoA _______________________ + NEM (2) Ratio 1:2 Whole homogenate Mitochondria 0.69 0.51 0.29 0.40 2.38 1.28 Sarcoplasmic reticulum 9.07 0.78 11.63
Subcellular fractions were obtained from homogenates of ventricle muscle. The values are expressed as nmol/min/mg of protein and are the means of 2 independent preparations.
40 \xM palmitoyl-CoA + 1 OmM NEM
40 pM oleoyl-CoA + 10 mM NEM
Mitochondria 0.39 0.02
120 |liM palmitoyl-CoA 120 pM oleoyl-CoA 120 pM oleoyl-CoA + 10 mM NEM
GPAT activity in heart SR, liver and WAT microsomal fractions was measured at several different concentrations of glycerol-3-phosphate (G3P). Eadie-Hofstee transformation of this data allowed determination of the apparent Km and Vmax of GPAT in these different tissues (fig 3.1). Expressed relative to protein, the Vmax o f GPAT in heart SR was similar to that of GPAT in liver microsomes, but was considerably lower than that of the WAT microsomal enzyme (table 3.3), which is unsurprising in view of the huge TAG synthesising capacity of the latter. In addition, the apparent Km of the SR GPAT for G3P was approximately 4-fold higher than that of the GPAT in WAT microsomes. The liver microsomal enzyme was quite similar to the heart enzyme in this respect. Whether these differences in Km reflect tissue-specific isoforms of GPAT or simply different microenvironments created by the reticular membranes o f each tissue is unclear. In either case, the difference may be physiologically relevant.
The G3P concentration in rat hearts perfused with glucose alone has been reported as 0.25 mM (Denton & Randle, 1967a), 0.14 mM (Trach et al, 1986) or 0.12 - 0.35 mM
(de Groot et al, 1989), rising to 0.6 - 0.9 mM with glucose plus lactate (de Groot et al,
1989), 0.57 mM with glucose plus insulin (Denton & Randle, 1967a), and 1.6 mM under ischaemic conditions (Trach et al, 1986). The cellular G3P concentration appears
to be somewhat higher in isolated cardiac myocytes and has been estimated at 1 - 2 mM (see section 3.3) or 4 mM (Myrmel et al, 1992), rising to as much as 12 mM under
prolonged hypoxia (Myrmel et al, 1991). Therefore, under most conditions it appears
that the G3P concentration in the heart will be considerably lower than the Km of the SR GPAT for this substrate. Since this is the major form of GPAT present in cardiac myocytes (see section 3.1.3), it is likely that cardiac TAG synthesis will be very responsive to factors which alter tissue levels of G3P. This may explain why TAG