Hepatic triacylglycerol lipase in circulating blood of normal and tumor-bearing mice and its hydrolysis of very-low-density lipoprotein and synthetic acylglycerols

A large amount of triacylglycerol lipase activity was present in the circulating blood of normal mice, and this activity decreased with development of Sarcoma 180 inoculated intraperitoneally. Triacylglycerol lipase in plasma of both normal and tumor-bearing mice was retained on the heparin-Sepharose columns and over 90% of the activity was eluted with 0.75 M NaCl. This enzyme had similar properties to hepatic triacylglycerol lipase and hydrolyzed very-low-density lipoprotein (VLDL)-triacylglycerol. Hepatic triacylglycerol lipase in plasma of normal mice hydrolyzed tricaprin and trilaurin most readily and better than 1-monoacylglycerols with the same acyl chain length. The hydrolyzing activities decreased with increase in the acyl chain length. The activity toward triolein was also higher than that toward 1-monoolein. 1-Monomyristin was hydrolyzed better than trimyristin. In contrast, hepatic triacylglycerol lipase in plasma of mice on day 4 after tumor inoculation hydrolyzed 1-monoacylglycerols better than triacylglycerols with the same acyl chain length. Hydrolysis of triolein by hepatic triacylglycerol lipase in plasma of both normal and tumor-bearing mice was reduced in the presence of 1-monoacylglycerols in the reaction mixture. The orders of their inhibitory effects coincided with the orders of the hydrolyzing activities toward 1-monoacylglycerols.     

Lipoprotein lipase activities in heart muscle of streptozotocin-induced diabetic rats

Triglyceride lipase (TGL) activities in the homogenates of the rat heart muscle were studied. TGL activity per mg protein of heart muscle was the highest in heart muscle homogenate utilizing 2.1 M glycine buffer, pH 8.3 among the assays investigated. The effects of NaCl, serum and heparin on TGL activities in heart muscle homogenates indicated the characteristics of lipoprotein lipase (LPL). Twelve-hour fasting increased heart muscle LPL activity, while enzyme activities in 48 hour- and 72 hour-fasted rats were lower than those in fed rats. LPL activities in heart muscle homogenates in streptozotocin (STZ)-induced diabetic rats either 3 days or 4 weeks after STZ injection, were decreased significantly as compared with those of control rats.     

Changes in ornithine decarboxylase activity in normal tissues of tumor-bearing mice during tumor growth.

The ornithine decarboxylase [EC 4.1.1.17] activities in the liver and spleen of tumor-bearing mice increased remarkably, reaching a peak 4 to 6 days after inoculation of tumor cells. On the contrary, the enzyme activity in the kidney decreased during tumor growth and had almost disappeared on day 6 after tumor inoculation. Injection of cell-free tumor homogenate also raised the enzyme activities in the liver and spleen, but did not change the activity in the kidney. No increase in enzyme activity in the liver of mice was observed on injection of homogenates of normal tissues, such as liver, spleen, kidney, and muscle.     

Further purification of human pituitary-derived chondrocyte growth factor: heparin-binding and cross-reactivity with antiserum to basic FGF

The previously described human pituitary-derived chondrocyte growth factor (CGF), mitogenic for rabbit fetal chondrocytes, was found to bind to heparin-Sepharose and was eluted with 1.5M NaCl. Further characterization of CGF demonstrated a molecular weight of 18-20 kD and cross-reactivity with antiserum to synthetic bovine basic fibroblast growth factor (FGF1-24). When human pituitaries were homogenized in 0.15 ammonium sulfate (pH 5.5) and the extract chromatographed on heparin-Sepharose, 98% of the mitogenic activity was adsorbed to heparin and eluted with 3M NaCl. These findings indicate that CGF is closely related or identical to basic FGF and that the bulk of mitogenic activity in the human pituitary extracts binds to heparin.     

Heparin-copper biaffinity chromatography of fibroblast growth factors

A novel method is described to separate and identify the various forms of fibroblast growth factor (FGF) based on their differential affinities for both heparin and copper. FGFs were extracted from bovine hypothalamus and purified by batchwise adsorption to heparin-Sepharose. The partially purified FGFs were then applied to an affinity column prepared by mixing equal portions of heparin-Sepharose and copper-Sepharose. The column was rinsed consecutively with the following four reagents: (i) 2 M NaCl, (ii) 0.6 M NaCl, (iii) 0.6 M NaCl plus 10 mM imidazole, and (iv) 0.6 m NaCl. FGFs were then eluted with a linear NaCl/imidazole gradient (from 0.6 m NaCl without imidazole to 2 M NaCl plus 10 mM imidazole). Fractions eluted from the column were analyzed by sodium dodecyl sulfate-gel electrophoresis with silver staining and electrophoretic immunoblot using site-specific antibodies against basic and acidic FGF. The results demonstrate that it is possible to resolve from hypothalamus at least two basic FGF species (with Mr values of 19,000 and 18,000) and three acidic FGF species (with Mr values of 18,000, 16,400, and 15,600). These findings indicate that heparin-copper biaffinity chromatography may have wide applicability in the study of the structure and activity of FGFs.     

Purification of rat adipose tissue lipoprotein lipase by affinity chromatography.

Lipoprotein lipase (EC 3.1.1.3) from rat adipose tissue was purified by affinity chromatography with heparin-Sepharose. Elution was carried out with buffered solutions of increasing NaCl molarity. Proteins without affinity for heparin were eluted with 0.5 M NaCl, while lipoprotein lipase activity was eluted as two peaks with 1.16 M NaCl (In earlier work on human adipose tissue (Etienne et al. (1974) C.R. Acad. Sc. Paris 279, 1487-1490) two fractions with lipoprotein lipase activity were also obtained). Phospholipase activity was detected in the fraction eluted with buffered 0.5 M NaCl and containing proteins without affinity for heparin. On feeding the fasting rats with fresh cream or glucose two peaks were also obtained, but the first peak had clearly increased while the second one had remained virtually unchanged.     

Characterization of triacylglycerol hydrolase activities in isolated myocardial cells from rat heart.

Triacylglycerol (TG) hydrolase activities were characterized in myocytes isolated from rat hearts. Acid hydrolase activity with a pH optimum of 5 could be measured in myocyte homogenates, and the subcellular distribution suggested that this activity originated in lysosomes. Lipoprotein lipase (LPL) was also present in myocyte homogenates, as evidenced by TG hydrolase activity that was stimulated by serum and apolipoprotein CII, and inhibited by apolipoprotein CIII2, high ionic strength (NaCl and MgCl2, I = 1 M) and antibodies to LPL. Serum-independent neutral (pH 7.5) TG hydrolase activity was less sensitive to inhibition by 1 M-NaCl, by antibodies to LPL and by preincubation at 40 degrees C than was serum-stimulated hydrolase activity. Furthermore, there were modest but significant differences in the subcellular distribution of the serum-independent and serum-stimulated hydrolase activities. Hydrolase activities in myocyte homogenates could be solubilized by 7.2 mM-deoxycholate. Acid hydrolase activity was recovered in the unbound fraction after heparin-Sepharose chromatography, whereas LPL was bound to the affinity column and was eluted by 0.9-1.2 M-NaCl. Approximately one-third of the serum-independent TG hydrolase activity was not bound to the heparin-Sepharose affinity column. This unbound TG hydrolase activity had a pH optimum of 7 and was stimulated by 50 mM-MgCl2, but not by serum and was resistant to inhibition by high ionic strength (1 M-NaCl), to preincubation at 40 degrees C for 2 h, and by antibodies to LPL. It is concluded that, in addition to an acid lysosomal TG hydrolase and LPL, myocytes from rat heart contain a serum-independent TG hydrolase with unique characteristics.     

Characterization of serum-stimulated lipoprotein lipase from bovine heart

1. A triglyceride (TG) lipase is present in whole homogenate and tissue extracts of beef myocardium with characteristics of lipoprotein lipase (LPL); i.e., activity is stimulated by serum, inhibited by NaCl and protamine sulfate, the protein binds to heparin-Sepharose, and the enzyme has an alkaline pH optimum. 2. This TG lipase, eluted from heparin-Sepharose at 0.9-1.0 M NaCl, has an apparent mol. wt of 64 K daltons. Its primary mRNA is 3.7 kb. 3. Expression of LPL mRNA and enzyme activities are in the ratio of approximately 20:8:1 for hearts of mouse, rat and beef, respectively and correlate with r = +0.99.     

A human prostatic growth factor (hPGF): partial purification and characterization

A growth factor capable of stimulating DNA synthesis of BALB/3T3 cells was purified about 1,000-fold from the cytosol of human benign hypertrophic prostates by heparin-Sepharose chromatography; the growth factor bound to the column in the presence of 0.5 M NaCl was eluted with 1.5-1.7 M NaCl. Its molecular weight and isoelectric point were estimated to be 11,000-13,000 and 10.5, respectively. It was sensitive to heat- and acid-treatments but resistant to disulfide-reducing agent. The final preparation was able to stimulate DNA synthesis at 10 ng/ml. The degree of stimulation was dependent on serum concentration in the assay system; the degree of maximum stimulation increased about 5 times as serum concentration increased from 0.2 to 2%.     

Heparin inhibits the activity of protein phosphatase-1

Heparin inhibited the dephosphorylation of rabbit skeletal muscle or liver phosphorylase a by protein phosphatase-1. Other glycosaminoglycans (chondroitin sulfates) and their constituents were found to be without effect. The chromatography of a partially purified phosphatase preparation on heparin-Sepharose CL-6B resulted in a fraction that did not bind to the matrix and its activity was not inhibited by heparin or inhibitor-1. The phosphatase bound to heparin-Sepharose was eluted by 0.2 M NaCl and was inhibited by heparin or inhibitor-1.     

The flowering of interferon

A microsomal vesicle fraction was prepared from rat liver homogenate by centrifugation in gradients of Percoll. The microsomes were subjected to gel filtration on Sephacryl S-1000 Superfine, which resolved the microsomes from Percoll. The elution pattern of the microsomal marker enzyme NADPH-cytochrome c reductase showed that the main part of the enzyme was present in a peak at Kav about 0.1, while Percoll eluted in a broad peak at Kav about 0.7. The total yield of eluted enzyme activity was 85%. The gel filtration had to be carried out in the presence of 10 mM tris or NaCl. At lower ionic strength or in 0.25 M sucrose alone, anomalous behaviour of the Percoll particles and microsomes on the gel was observed. Electron microscopy of samples from the void volume fraction of the Sephacryl S-1000 Superfine column showed an almost complete removal of Percoll from the microsomes. Furthermore, the vesicle preparation was essentially free of membrane fragments.     

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with the non-ionic detergent Nonidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaCl-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaCl from the DNA-cellulose column, P5 and P5′ are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaCl eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaCl, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.     

Identification of the hepatocyte mitogen in bovine spleen as heparin-binding growth factors.

Growth promoting activity for rat hepatocytes in bovine spleen was identified as three heparin-binding growth factors. All the features tested, such as heparin affinity, molecular mass, cross reactivity with antibody, and partial amino acid sequence, indicated that one of the three factors was identical to FGF-1 (fibroblast growth factor-1, acidic FGF), another one was related to FGF-2 (fibroblast growth factor-2, basic FGF), whereas it was more potent for hepatocytes than the FGF-2 purified from bovine brain. The third one was eluted from heparin-Sepharose column at 0.75M NaCl, of which activity was not abolished by anti-FGF-1 or FGF-2 antibodies. In addition, the mitogenic effect of this factor was synergistic with that of HGF (hepatocyte growth factor), a known potent hepatocyte mitogen, suggesting that it is a novel growth factor for hepatocytes.     

Increased content of calmodulin in morris hepatoma 5123 t.c.(h)

The content of calmodulin in the 100,000 × g supernatant and particulate fractions in Morris hepatoma 5123 t.c.(h), assayed by its ability to activate the Ca²⁺-activatable cAMP phosphodiesterase, was significantly higher (about 44%) than that in normal or host liver. Only one peak of calmodulin activity was detected when 100,000 × g supernates (350 mg protein) from sonicated homogenates of normal liver and hepatoma were fractionated on a DEAE cellulose column, eluting at a sodium acetate concentration of 0.65 M. The total calmodulin activity eluted from the hepatoma supernatant was 240% higher than that from nornal liver. The increased content of calmodulin in the hepatoma may contribute to the state of abnormal cell proliferation in this tissue.     

Synthesis of lipoprotein lipase in cultured avian granulosa cells

Avian granulosa cells cultured as a homogeneous parenchymal population contain lipolytic activity. This activity is stimulated 2--5-fold by serum, inhibited 90% by 1 M NaCl and inhibited 80% by specific anti-lipoprotein lipase immunoglobulins. 85% of the activity binds to heparin-Sepharose 4B, and 70% of bound activity is eluted with 1.5 M NaCl. Thus, the lipolytic activity of cultured granulosa cells is lipoprotein lipase. Granulosa cells were shown to synthesize lipoprotein lipase in culture by incorporating [3H]leucine into the enzyme protein, as measured with an immunoadsorption technique. Finally, colchicine was shown to increase intracellular lipolytic activity, suggesting an inhibition of secretion of this enzyme by cultured granulosa cells.     

Affinity chromatography of embryonic inducing factors on heparin-Sepharose

Mesoderm-inducing factors were extracted from chicken embryos and partially purified by chromatography on DEAE-cellulose. The DEAE-cellulose eluate was applied to heparin-Sepharose. Most of the proteins are not bound to heparin. The adsorbed proteins were eluted with a linear NaCl gradient. In totipotent gastrula ectoderm of amphibians the eluted proteins induce the differentiation of muscle and notochord as well as of large masses of renal tubules and blood cells. A possible relationship to fibroblast growth factors and angiogenesis factor is discussed.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Heparin-released triglyceride lipase from chang liver cells

Heparin-released triglyceride lipase (TGL) from Chang liver cells (ATCC CCL 13) was investigated using very low density lipoproteins (VLDL) as a substrate. The TGL activity was released into the culture medium when the cells were incubated with heparin. The enzyme showed maximum activity at pH 8.5, and 80% inhibition by 0.6 M NaCl. These results indicated that Chang liver cells, a cell line derived from liver, synthesize lipoprotein lipase.     

Purification of the neurotensin receptor from mouse brain by affinity chromatography

The neurotensin receptor was purified from newborn mouse brain by affinity chromatography. Active neurotensin binding sites were solubilized from brain homogenates using the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in the presence of cholesteryl hemisuccinate. Chromatography of the soluble extract on SP-Sephadex C-25 and hydroxylapatite eliminated 50% of proteins without loss of neurotensin binding activity. This prepurified material was loaded into an affinity column prepared by coupling neurotensin (2-13) to glutaraldehyde-activated Ultrogel AcA22. Nonspecifically adsorbed proteins were eliminated by extensive washing, and the receptor was eluted with a buffer containing 1 M NaCl, 0.1% CHAPS, and 0.02% cholesteryl hemisuccinate. After desalting, the purified receptor bound 125I-labeled neurotensin with a dissociation constant of 0.26 nM and retained its specificity towards a series of neurotensin analogues. The desalted NaCl eluate appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major band of molecular weight 100,000 which was identified as the receptor by affinity labeling with 125I-labeled neurotensin in the presence of disuccinimidyl suberate. The purity of the mouse brain receptor eluted from the affinity column was estimated to be 78%. Electroelution of the 100-kDa protein band gave an homogenous preparation of receptor. Very similar results were obtained with CHAPS-solubilized neurotensin receptors from rat and rabbit brain.     

Isolation and properties of triglyceride lipase from the rabbit liver

Using affinity chromatography on heparin-Sepharose 4B, triglyceride lipase was isolated from rabbit liver tissue and purified. The specific activity of the enzyme isolated from the usual homogenate was equal to (3.8 +/- 1.2) x 10(3) mumol/hour/mg protein. After treatment of liver tissue homogenates with liquid nitrogen the enzyme activity increased severalfold as compared to the enzyme isolated from the usual homogenate. The dependences of the triglyceride lipase activity on the concentrations of the protein (enzyme), substrate (triglyceride), albumin (fatty acid acceptor) and pH were studied. The isolated form of liver triglyceride lipase was found to have two pH optima at 6.5 and 8.5.