Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

Appraisal of hepatic lipase and lipoprotein lipase activities in mice

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.     

脂肪酶基因在枯草芽孢杆菌中的表达及表达产物性质的研究

从环境中筛选到了脂肪酶高产菌株金黄色葡萄球菌JH,依据NCBI上发表的原核微生物脂肪酶基因序列的多序列比对,发现它具有很强的序列保守性。利用PCR从金黄色葡萄球菌JH基因组中扩增得到了脂肪酶基因,利用基因重组技术将其整合到质粒pC194中,并导入到枯草芽孢杆菌中进行表达。应用选择抗药性筛选重组子,利用硫酸铵沉淀法和离子交换层析分离纯化重组脂肪酶,并用DS-PAGE进行纯度鉴定,确定其相对分子量约为32000Da。通过对其酶学特性的研究发现,重组脂肪酶在反应温度为41℃, pH为8.0时具有最大活性,其Km和Vm各自为0.34mM和308μmol•mg-1min-1,Ca2+、K+、Mg2+能激活这种酶的活性鳩e2+、Cu2+、Co2+则抑制它的活性。     

Lipoprotein lipase and acid lipase activity in rabbit brain microvessels.

A preparation of cerebral microvessels was used to demonstrate the presence of lipoprotein lipase and acid lipase activity in the microvasculature of rabbit brain. Microvessels, consisting predominantly of capillaries, small arterioles, and venules, were islated from rabbit brain. Homogenates were assayed for lipolytic activity using a glycerol-stabilized trioleoylglycerol-phospholipid emulsion as substrate. Lipoprotein lipase activity was characterized with this substrate by previously established criteria including an alkaline pH optimum, increased activity in the presence of heparin and heat-inactivated plasma, and reduced activity in the presence of NaCl and protamine sulfate. A different substrate, containing trioleoylglycerol incorporated into phospholipid vesicles, was used to reveal acid lipase activity that was not affected by heparin, plasma, NaCl, or protamine sulfate. Lipoprotein lipase did not show activity with the vesicle preparation as substrate. Intact microvessels, when incubated in the presence of heparin, release lipoprotein lipase into the incubation solution. In contrast, release of acid lipase activity from intact microvessels was not dependent on heparin. The data show the presence of both lipoprotein lipase and acid lipase in brain microvessels and suggest that lipoproteins are metabolized within the cerebral vasculature.     

Compound heterozygosity for mutant hepatic lipase in familial hepatic lipase deficiency

In a kindred with three hyperlipidemic subjects who had premature atherosclerosis and complete deficiency of hepatic lipase activity, we had previously identified a novel structural hepatic lipase gene variant. We now report the identification of three more hepatic lipase gene mutations in this family and demonstrate that compound heterozygosity for two hepatic lipase mutations (designated S267F and T383M) underlies hepatic lipase deficiency.     

The N-terminal His-tag affects the triglyceride lipase activity of hormone-sensitive lipase in testis

The sterility of hormone-sensitive lipase (HSL) knockout mice clearly shows the link between lipid metabolism and spermatogenesis. However, which substrate or product of this multifunctional lipase affects spermatogenesis is unclear. We found that an HSL protein with a His-tag at the N-terminus preserved the normal hydrolase activity of cholesteryl ester (CE) but the triglyceride lipase (TG) activity significantly decreased in vitro. Therefore, mice with this functionally incomplete HSL (His-HSL) were produced on a background of HSL deficiency (HSL^−/−h). As a result, HSL^−/−h testis has an 8.65-fold higher CE activity than wild-type testis but a twofold higher TG activity than wild-type testis. To compare His-HSL and wild-type HSL in vitro and in vivo, we confirmed that the His-tag significantly suppressed HSL TG activity. From our results, we believe that TG activity was affected by the His-tag insertion, but CE activity was not influenced. Furthermore, the His-tag protected HSL from binding to the inhibitor BAY. From our study, TG activity and BAY binding sites were affected by N-terminal His-tag insertion.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

Development of lipase in nursing piglets

This experiment was designed to investigate the development of gastric lipase and pancreatic lipase in nursing piglets. During the nursing period, the lipase activity was measured at one, 7, 14, 21 and 28 days of age. The results showed that the gastric mucosa weight, pancreas weight, body weight, the specific activity of gastric lipase and pancreatic lipase, and the total activity of gastric lipase and pancreatic lipase increased with the age of the piglets. The development of the specific activity of gastric lipase slowed before the nursing piglets reached 3 weeks of age, but the total activity of gastric lipase at day 28 was significantly higher than that at day 21. The specific activity and total activity of pancreatic lipase were at low levels during the first two weeks of life and then developed quickly from days 21 to 28. It was observed that the specific activity and total activity of gastric lipase were lower than those of pancreatic lipase.     

Endothelial lipase: a new lipase on the block

Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.     

Triacylglycerol lipase in bovine erythrocytes

Acid lipase activity was found in the bovine erythrocyte ghosts, but little neutral or alkaline lipase activity was observed in the erythrocytes. The membrane-bound lipase showed a remarkable activity in the ghosts only after hemolysis. The membrane-bound lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The hydrolysis rate was linear with time up to 60 min, and was proportional to the amount of enzyme up to 0.4 mg protein. The bound lipase was activated markedly by bovine serum albumin and slightly by octyl-glycoside. The lipase was remarkably inhibited by bovine serum.