A serum protein inhibitor of acid lipase and its possible role in lipid accumulation in cultured fibroblasts.

Histochemical examination of L929 fibroblasts indicates massive accumulation of intracellular lipids in cells grown in medium supplemented with 10% calf serum. The present study suggests that the accumulation of triacylglycerols in these cells may be due to the inhibition of acid lipase activity by a serum component present in the culture medium. This is based on the following observations. (a) Acid lipase appears to be the major intracellular enzyme responsible for triacylglycerol catabolism in L929 cells. (b) The acid lipase is strongly inhibited by either human of calf serum. Several lines of evidence show that the inhibitor is a serum protein: it is heat-labile, non-dialysable and is destroyed by trypsin. It is present mainly in Cohn's fraction IV and has mol.wt. approx. 50000. (c) Lipid accumulation in intact cells is reduced when cells are grown on a limited supply of serum (2%) and is elevated by the addition of Cohn's fraction IV, freed of lipoproteins, to the growth medium.     

Fasting-induced inhibition of collagen biosynthesis in rat skin. A possible role for phosphoenolpyruvate in this process

Fasting is accompanied by a decrease in collagen biosynthesis. The mechanism of this phenomenon involves inhibition of prolidase activity, an enzyme that plays a key role in upregulation of collagen metabolism. The mechanism of fasting-induced inhibition of prolidase activity is not known. Phosphoenolpyruvate (PEP) is known as a strong inhibitor of prolidase activity. It exerts this effect by inhibition of the enzyme phosphorylation. Unphosphorylated prolidase is inactive. One may expect that fasting-associated increase in posphoenolpyruvate content in animal tissues may be a factor which inactivates prolidase and makes it inactive in collagen biosynthesis. We measured the levels of phosphoenolpyruvate, pyruvate, and pyruvate kinase in the skin of control and fasted rats and correlated these parameters with prolidase expression, prolidase activity and collagen biosynthesis in this tissue. Significant increase of PEP concentration (about 30%) was found in the skin of fasted rats. In the same time prolidase activity and collagen biosynthesis decreased by about 50% and 30%, respectively, compared to controls. It is known that phosphoenolpyruvate is converted to pyruvate by the action of pyruvate kinase. Since fasting significantly decreases the activity of this enzyme, one may suggest that the accumulation of PEP is caused by a reduced utilisation of this metabolite. As demonstrated by Western immunoblot analysis the decrease in prolidase activity was not accompanied by a decrease in the amount of the enzyme protein. Instead, a decrease in the enzyme phosphorylation was observed. The reduction in phosphorylation seems to be responsible for the decrease in prolidase activity. These data suggest that fasting-evoked accumulation of PEP reduces the activity of prolidase, providing a mechanism for inhibition of collagen biosynthesis in the skin.     

Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism

Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantly at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMP-dependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo.     

A possible role for melatonin in regulation of chick liver xanthine dehydrogenase activity.

³⁵Cl nuclear magnetic resonance longitudinal and transverse relaxation times were employed to study anion binding to rabbit muscle lactate dehydrogenase. The correlation time, obtained from a comparison of the two relaxation times, shows that coenzyme has a marked retarding effect on the anion mobility at the binding site. The quadrupole coupling constant is estimated from the magnitude of the relaxation rate change on oxamate addition.     

Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.

The thermodynamic stability of beta-lactoglobulin (beta-Lg) was studied at acidic and near-neutral pH values using equilibrium thermal-unfolding measurements. Transition temperature increased with a decrease in pH from 7.5 to 6.5 and 3.0 to 1.5, suggesting an increase in the net protein stability. Determination of the change in free energy of unfolding and extrapolation into the nontransition region revealed that beta-Lg increases its stability by increasing the magnitude of the change in free energy of unfolding at the temperature of maximum stability, as well as by increasing the temperature of maximum stability. The relative difference in the change in free energy of unfolding at 70 degrees C (with a reference pH of 7.5) was positive and its magnitude increased with a decrease in pH from 7.0 to 1.5 van't Hoff plots of thermal unfolding of beta-Lg at all pH values studied were non-linear and the measured changes in the enthalpy and entropy of unfolding for beta-Lg were high and positive. The relative magnitude of change of both enthalpy and entropy at 70 degrees C (compared with pH 7.5) increased with a decrease in pH up to 1.5. A possible mechanism for the increased stability of beta-Lg at low pH is discussed.     

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.     

Molecular dynamics simulations of the whey protein beta-lactoglobulin.

Molecular dynamics simulations have been used to model the motions and conformational behavior of the whey protein bovine beta-lactoglobulin. Simulations were performed for the protein by itself and complexed to a single retinol ligand located in a putative interior binding pocket. In the absence of the retinol ligand, the backbone loops around the opening of this interior pocket shifted inward to partially close off this cavity, similar to the shifts observed in previously reported molecular dynamics simulations of the uncomplexed form of the homologous retinol binding protein. The protein complexed with retinol does not exhibit the same conformational shifts. Conformational changes of this type could serve as a recognition signal allowing in vivo discrimination between the free and retinol complexed forms of the beta-lactoglobulin molecule. The unusual bending of the single alpha-helix observed in the simulations of retinol binding protein were not observed in the present calculations.     

Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans.

Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.     

Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.     

Clearing-factor lipase in adipose tissue. Distinction of different states of the enzyme and the possible role of the fat cell in the maintenance of tissue activity

1. Incubation of intact epididymal adipose tissue from fed rats at 37 degrees in an albumin solution at pH7.4 in vitro results in rapid loss of clearing-factor lipase activity until a low activity, stable to prolonged incubation, is attained. The clearing-factor lipase activity of intact tissue from starved rats, which is initially much less than that of tissue from fed rats, is mainly stable to incubation at 37 degrees . 2. Much of the clearing-factor lipase activity of intact epididymal adipose tissue from fed rats is inactivated by collagenase. The enzyme activity of intact tissue from starved rats is not inactivated by collagenase. 3. The clearing-factor lipase activity of fat cells isolated from the epididymal adipose tissue of fed rats is stable to prolonged incubation at 37 degrees . It represents only a small proportion of the total activity of the intact tissue. In starved rats, the isolated fat cells contain a much higher proportion of the activity of the intact tissue. Their activity is also stable at 37 degrees . 4. Incubation of isolated fat cells in a serum-based medium leads to a progressive rise in clearing-factor lipase activity. Actinomycin increases the extent of this rise in activity. No rise in clearing-factor lipase activity occurs when stromal-vascular cells isolated from epididymal adipose tissue are incubated in the medium. 5. The findings indicate that less than 20% of the activity of intact adipose tissue from fed rats is retained when fat cells are isolated from the tissue by collagenase treatment. The activity that is lost could be that which normally functions in the uptake of triglyceride fatty acids by the tissue.     

Enzymatic activity of protein kinase LOSK: possible regulatory role of the structural domain

LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis. LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis. To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E. coli. Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable. H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides. The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain. The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity. Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone. Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect. Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase. These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.