Bovine brain microsomal CDP-diacylglycerol synthetase: solubilization and properties.

CDP-diacylglycerol(DAG) synthetase (EC 2.7.7.41) has been solubilized from bovine brain microsomes by the detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate). Optimal solubilization with 1.5% CHAPS yielded 55-60% of the synthetase activity. The effect of CHAPS on the enzyme was biphasic inhibiting at 0.3% and giving maximal activity at 0.5% (the concentration used for all assays). The solubilized, but not the microsomal enzyme is activated by phosphatidylcholine (PC) and strongly inhibited by cardiolipin and lysoPC. Strong inhibition by N-ethylmaleimide, 5,5'-dithio-bis (2-nitrobenzoic acid) and p-chloromercuribenzoate supported a sulfhydryl requirement for the enzyme. Phosphatidic acid (PA) from egg lecithin and 1-stearoyl,2-arachidonoyl PA were preferred substrates for the microsomal synthetase. Solubilized synthetase showed selectivity for the latter PA which is consistent with this enzyme functioning to help form the preponderant 1-stearoyl,2-arachidonoyl species of phosphatidylinositol. Further attempts to purify the synthetase were unsuccessful. All findings suggested the enzyme exists as an unstable complex.

Triacylglycerol synthesis and diacylglycerol acyltransferase activity during skeletal myogenesis.

The role that diacylglycerol acyltransferase (DAGAT) may play in the switch in lipid metabolism from predominantly triacylglycerol- and phospholipid-synthesizing myoblasts to predominantly phospholipid-synthesizing myotubes has been studied during L6 skeletal myogenesis. Fatty acid induced triacylglycerol (TAG gamma accumulation in vivo was found to be optimal with long-chain, unsaturated fatty acids. The fatty acid induced TAG accumulation was significantly greater in myoblasts than that in myotubes. DAGAT activity in vitro was found to be associated with the particulate (membrane) fraction only. The inhibition by many thiol-specific reagents (N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) suggest that a thiol group is at or near the active site. In general, optimal DAGAT activity in vitro was observed when long-chain unsaturated acyl-CoAs and diacylglycerols (DAGs) containing long acyl chains were used as substrates for in vitro TAG synthesis (although 1,2-didecanoin was also very effective). DAGAT activity (expressed relative to DNA) was shown to decline over twofold during skeletal myogenesis when measured in the absence of exogenous DAG. However, in the presence of exogenous (1 mM) DAG, there was no significant change in DAGAT activity, suggesting that the levels of this enzyme are not altered during skeletal myogenesis. These results indicate that endogenous DAG levels are limiting TAG synthesis in L6 myotubes. However, DAG content of myotubes was significantly greater than that of myoblasts, suggesting that there may be an increased competition for DAG (perhaps owing to enhanced phospholipid synthesis) during skeletal myogenesis. The combined effects of decreased synthesis and increased degradation (reported earlier) of TAG may account for the decrease in endogenous TAG contents observed during skeletal myogenesis.

Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters.

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extract and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.

The purification and properties of monoacylglycerol kinase from bovine brain.

Monoacylglycerol kinase (MGK) has been purified from bovine brain by six steps: isolation of cytosol, DEAE-cellulose chromatography, ammonium sulfate fractionation (0-40%), Bio-Gel A-1.5m, hydroxylapatite, and ATP-agarose column chromatography. The overall purification was 938 times with a 4.8% yield. The column separations (particularly Bio-Gel A-1.5m) and SDS- and nondenaturing-polyacrylamide gel electrophoresis of enzyme purified from ATP-agarose indicated that MGK exists as a complex (approximately 350 kilodaltons) that is stabilized by 0.5 M NaCl and, on complete dissociation, yields a major protein of 72 kilodaltons. Dithiothreitol, EDTA, and ATP helped to stabilize MGK during purification. The protein peak eluted from hydroxylapatite by 25 mM phosphate activated and stabilized MGK activity. Phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin inhibited MGK. These phospholipids and others activated MGK synergistically with the above protein peak. MGK copurified with diacylglycerol kinase (DGK) throughout giving MGK to DGK ratios of 0.05-0.36. Optimal activity required 0.5 mM 2-monoolein and 10 mM MgCl2. Strong inhibition by p-chloromercuriphenyl sulfonic acid, N-ethyl-maleimide, and 5,5'-dithio-bis(2-nitrobenzoic acid), and prevention of this inhibition by dithiothreitol indicated the involvement of intact SH groups in the action of MGK. Purified MGK showed preference for substrates with unsaturated fatty acids except for 1- or 2-monostearin. Overall the preference favored the selective generation of 1-stearoyl- and 2-arachidonoyl-lysophosphatidic acid.

Changes in phospholipid metabolism dependent on calcium-regulated myoblast fusion.

Fusion-competent myoblasts can be prevented from fusing (differentiating) by reducing medium calcium concentrations from 1.65 mM to less than 50 microM. Fusion is completely retarded after 24 h but is noticeable after 48 h and significant after 72 h in low-calcium medium. After 24 h in low-calcium medium, a rapid, synchronous fusion can be initiated by return to normal (high-calcium) medium. Calcium content increases over threefold during myoblast differentiation and closely parallels the fusion process. Phospholipid content is also dependent upon the state of differentiation. Myotubes (fused myoblasts) have an almost twofold greater content of lipid phosphate per milligram of protein compared with that of myoblasts; this increase is localized to increased contents of phosphatidylcholine and pooled phosphatidylinositol - phosphatidylserine. Phospholipid synthesis (32Pi incorporation) is markedly stimulated four- to five-fold when myoblasts grown in low-calcium medium are switched to normal medium. These significant increases are observed in all the major phospholipids studied, predominantly in phosphatidylcholine and pooled phosphatidylinositol - phosphatidylserine, and most noticeably in phosphatidylinositol 4,5-bisphosphate. Furthermore, we show that phosphatidylinositol 4,5-bisphosphate prelabelled with myo-[2-3H]inositol is rapidly degraded after switching from low-calcium medium to normal medium. These changes are not observed in myotubes treated similarly, which suggests that the changes in phospholipid metabolism may be fusion related. These results support a proposal by another author, which suggests that phosphatidylinositol 4,5-bisphosphate breakdown may play an important regulating role in myoblast differentiation.

Changes in oleic acid oxidation and incorporation into lipids of differentiating L6 myoblasts cultured in normal or fatty acid-supplemented growth medium.

L6 myoblasts accumulate large stores of neutral lipid (predominantly triacylglycerol) when cultured in fatty acid-supplemented growth medium. No accumulation of neutral lipid was evident in myotubes (differentiated myoblasts) when treated similarly. Triacylglycerol accumulation was rapid and dependent on exogenous fatty acid concentration. Triacylglycerol content in myoblasts cultured in fatty acid-supplemented growth medium was approx. 3-fold higher than that in myotubes treated similarly and 2-3-fold higher than that in myoblasts cultured in normal growth medium. Incorporation studies using [I-14C]oleic acid showed that myoblasts and myotubes take up exogenous fatty acid at similar rates. However, cells cultured in fatty acid-supplemented growth medium remove more exogenous fatty acid than do cells cultured in normal growth medium. Over 90% of the incorporated label was found in phospholipid and triacylglycerol fractions in all situations studied. Myoblasts incorporated a more significant proportion (P less than 0.001) of label into triacylglycerol compared with that of myotubes. No differences in fatty acid oxidation rates were detected when differentiating L6 cells cultured in normal growth medium were compared with those cultured in fatty acid-supplemented growth medium. However, fatty acid oxidation rates were observed to increase 3-5-fold upon myoblast differentiation. We conclude that there is a marked change in the pattern of lipid metabolism when myoblasts (primarily triacylglycerol-synthesizing cells) differentiate into myotubes (primarily phospholipid-synthesizing cells). Understanding these changes, which coincide with normal muscle development, may be important, since a defect in this natural switch could explain the observed accumulation of lipid in muscle characteristic of some of the muscular dystrophies and other lipid-storage myopathies.

Calmodulin levels in developing muscle tissues and primary cultures of normal and dystrophic (UM-X7.1) hamsters.

Calmodulin levels have been assessed in whole muscle and primary culture extracts in order to examine the relationship between calmodulin and the accumulation of calcium in dystrophic hamster muscle tissues. Significant decreases in both normal and dystrophic skeletal muscle, tongue, and heart calmodulin levels were observed between 2 and 12 weeks of age. Dystrophic values, however, tended to be somewhat higher than normal, especially in 12-week-old skeletal muscle total and soluble extracts (normal 29.7 and 0.6 and dystrophic 117.0 and 3.1 micrograms/g wet weight, respectively). No significant differences were observed in dystrophic myoblast (total 2.22-2.78, soluble 2.85-3.26 micrograms/mg protein) or fibroblast (total 2.64-2.94, soluble 2.54-3.60 micrograms/mg protein) calmodulin levels, except for a significant decrease in dystrophic fibroblast levels (total 1.97, soluble 2.18 micrograms/mg protein) at 7 days in culture. Elevated calmodulin levels in dystrophic muscle are discussed in terms of increases in intracellular Ca2+ concentrations and immature regenerating fibers.

Normal and dystrophic hamster myoblast and fibroblast growth in culture.

Normal and dystrophic hamster myoblasts and fibroblasts were compared for characteristic indicators of growth and differentiation. Clonal analysis of myoblast cultures indicated that 80% of colonies judged to be fusion-competent had differentiated. Dystrophic myoblasts were identical to normal in terms of their morphology, fusion potential (81.4%), and myokinase activity (59.6-49.1 mU/mg at 2-7 days), but displayed a significantly higher plating efficiency (normal: 52.6%; dystrophic: 82.1%), a longer doubling time (normal: 21.7 hours; dystrophic: 33.3 hours), and a lower day-7 creatine kinase activity (normal: 60.7 mU/mg; dystrophic: 41.3 mU/mg). Dystrophic fibroblasts were indistinguishable from normal ones in terms of their morphology, plating efficiency (90.4%), and doubling time (32.5 hours), but displayed a significantly lower day-2 creatine kinase activity (normal 58.3 mU/mg; dystrophic: 35.8 mU/mg) and day-7 myokinase activity (normal: 52.7 mU/mg; dystrophic: 39.9 mU/mg). The results are suggestive of an early and differential expression of the primary defect in dystrophic hamster myoblasts and fibroblasts in culture.

Properties of diacylglycerol kinase purified from bovine brain.

A nearly homogeneous but somewhat unstable diacylglycerol kinase (ca. MW 72,000 daltons) was purified from bovine brain by modification of the procedure of Kanoh et al. (Kanoh, H., Kondoh, H., and Ono, T. [1983] J. Biol. Chem. 258, 1767-1774). The purification consisted of four steps (brain cytosol isolation and successive chromatography on DEAE-cellulose, Sephadex G-25 for desalting and ATP-agarose) carried out in buffers stabilized with EDTA, ATP and dithiothreitol (DTT). Specific activities, determined within 4 hr of purification, ranged from 908-1857 nmol ATP incorporated/min/mg protein, with the variation reflecting the instability. Optimal activities required deoxycholate (0.1%), one of the phosphoglycerides [phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS)] (0.025-0.25 mM), ATP (5 mM, apparent Km = 0.57 mM), 1,2-dioleoyl-rac-glycerol (5 mM, apparent Km = 1 mM) and Mg2+ (10 mM, apparent Km = 2.2 mM). Phosphatidylinositol (PI) was slightly less effective than PC, PE or PS and noninhibitory in combination with PC, PE or PS. Relative to PC phosphatidic acid (PA) (52%), sphingomyelin (48%), lyso-PC (1.5%) and lyso-PI (28.6%) were less effective activators. The sulfhydryl reagents, p-chloromercuribenzoic acid (PCMB) (1.0 mM), N-ethylmaleimide (NEM) (1.0 and 2.0 mM) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (1.0 mM), showed strong inhibition of activity which was prevented by 0.5 mM DTT. In contrast to other reports, this purified enzyme showed no monoacylglycerol kinase activity. Comparison of diacylglycerols of varying fatty acid composition indicated that the enzyme showed a preference for substrates with at least one unsaturated fatty acid, particularly in the 2-position.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysosomal triacylglycerol lipase activity in L6 myoblasts and its changes on differentiation.

L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver.

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20-40 microM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosyl-methionine from ATP and methionine.

Calmodulin content and Ca-activated protease activity in dystrophic hamster muscles.

As part of a study on the implication of elevated Ca2+ levels in the myofibrillar degeneration seen in dystrophic muscle, the content of calmodulin and the activity of Ca2+-activated neutral protease (CANP) have been measured in normal and dystrophic (UM-X7.1) hamsters. Calmodulin levels, expressed as micrograms +/- SEM per gram wet weight were highest in brain (385 +/- 24.7), followed by tongue (93.88 +/- 3.93), heart (42.13 +/- 2.93), and skeletal muscle (31.69 +/- 1.42). No significant increases in calmodulin were observed in the dystrophic tissues thus suggesting that the Ca2+ accumulations observed in dystrophic muscles are unrelated to changes in a calmodulin levels. Because of the complexity of regulation of CANP, a time-dependent study was done using extracts of skeletal, heart, and tongue muscles. Marginal increases in dystrophic CANP were seen in skeletal muscle at all times studied and in the heart and tongue at initial time points only. The data are discussed in terms of rising levels of Ca2+ in muscles of the UM-X7.1 hamster being sufficient to increase CANP activity (without increasing content) to where it causes Z-line dissolution.

Insulin binding to myotonic dystrophy fibroblasts.

Insulin receptor binding was examined in cultured skin fibroblasts from 10 myotonic dystrophy patients and 10 age- and sex-matched control subjects. The conditions for insulin binding to fibroblasts were optimal and employed HEPES binding buffer, pH 8.0 at 15 degrees C for 5 h. These conditions correspond to those previously employed with monocytes from MyD subjects. The normalized initial insulin binding capacity showed a decrease of 62% from 5.04 +/- 0.28% of the total labeled insulin added/mg protein in the control to 1.93 +/- 0.13% in the myotonic dystrophy group (P less than 0.01) due mainly to a marked reduction in high affinity receptors or in receptor affinity. The addition of 1.0 ng/ml of unlabeled insulin produced significant decreases to 3.80 +/- 0.25% in the control group and 1.24 +/- 0.09% in the MyD group. The results are similar to previously reported findings with monocytes from myotonic dystrophy patients and suggest that a surface membrane defect exists in this disease. However, the conditions that have been employed in the binding procedures in all of the studies, while optimal, are performed at a high pH and low temperature and could have an important bearing on the interpretation of a membrane disorder.

On the specificity of a phospholipase A2 purified from the 106,000 X g pellet of bovine brain.

Assessment has been made of the specificity of a purified phospholipase A2 from the 106,000 X g pellet (microsomal fraction) of bovine grey matter which shows strong activity toward phosphatidylinositol (PI). In the first series of experiments involving the utilization as substrates of PI with different 14C- or 3H-labeled fatty acids in the 2-position, the purified phospholipase A2 most readily removed 16:0 palmitic acid, followed by 18:0 stearic acid, 18:1 oleic acid and 20:4 arachidonic acid. In the second series of experiments, the purified phospholipase A2 showed preferential action toward PI (100%) compared to phosphatidylcholine (PC, 62.5%), phosphatidic acid (PA, 32.6%), phosphatidylethanolamine (PE, 25.1%) and phosphatidylserine (PS, 21.5%), where each phosphoglyceride was labeled in the 2-position with [1-14C] oleic acid. In the third series of experiments, fatty acids were shown to cause inhibition of action of the purified phospholipase A2 on 1-acyl, 2-[1-14C] oleoyl PI in the order 20:4 greater than 18:1 greater than 18:0 greater than 16:0 which is the reverse order to that just noted. In the final series of experiments, the addition of the phosphoglycerides PC, PE, PS and PA in amounts of 5 or 10 microM caused either no inhibition (PE, 2%), slight inhibition (PC, 15%) or reasonably significant inhibition (PA, 20% and PS, 40%) of action of the purified phospholipase A2 on 1-acyl, 2-[1-14C]-oleoyl PI. The pattern of specificity observed for the purified phospholipase A2 combined with its microsomal location are the expected properties of a phospholipase A2 that might function in a deacylation-reacylation cycle for modifying the fatty acid distribution in PI.

Glucose transport and oxidation in adipose tissue of patients with myotonic dystrophy.

The effect of insulin on the transport of 2-deoxyglucose and the oxidation of glucose in chopped adipose tissue was investigated in 14 myotonic dystrophy (MyD) patients and 28 age and size-matched control subjects. The transport of 0.55 mM 2-deoxyglucose was measured over 3 min at 37 degrees C both with and without 32 ng/ml of insulin. Oxidation was determined at 37 degrees C for 90 min by the measurement of 14CO2 released from a system containing 0.55 mM glucose with and without 50 ng/ml of insulin. Basal 2-deoxyglucose transport was not reduced in MyD subjects but insulin-stimulated 2-deoxyglucose transport in MyD was significantly less at 0.512 +/- 0.220 nmole compared to control subjects with 0.906 +/- 0.160 nmole/100 mg tissue/3 min (P less than 0.02). Both the basal and insulin-stimulated glucose oxidation were significantly less in the MyD group. Insulin-stimulated oxidation was 2.92 +/- 0.21 nmole in the control subjects compared to 2.20 +/- 0.27 nmole/100 mg tissue/90 min in the MyD cases (P less than 0.02). Similar findings were obtained when calculations were based on nmoles of 2-deoxyglucose transport and glucose oxidation/100 mg lipid. The findings indicate that both glucose transport and oxidation are impaired in MyD.

On the ultrastructure of primary cultures of normal and dystrophic hamster tongue muscle.

Primary cultures, prepared from the tongue muscle of 1-day-old normal and dystrophic (UM-X7.1 strain) hamsters, were examined under the electron microscope after 6 to 8 days in vitro when myotube development was in progress. Myotubes from normal tongue showed development similar to that of muscle of other tissues and species grown in culture. Compared to myotubes of normal cultures, those of dystrophic cultures showed a number of differences. The sarcoplasmic reticulum and mitochondria showed areas of dilation or swelling and poor development. The density of ribosomes and polyribosomes was less, Z-band formation was primitive or absent, myofibrillar formation was less, and its organization was poor. Also, spontaneous contractions in dystrophic myotubes were few and irregular. These differences agree well with those reported for heart cells from the dystrophic (cardiomyopathic) hamster grown in culture.