John A. Glomset

Prenyl proteins in eukaryotic cells: a new type of membrane anchor

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

The Metabolic Role of Lecithin: Cholesterol Acyltransferase: Perspectives from Pathology

Publisher Summary This chapter discusses the metabolic role of enzyme lecithin called cholesterol acyltransferase (LCAT). The role of the LCAT reaction is to prevent unesterified cholesterol, derived mainly from the surfaces of chylomicrons and very low density lipoproteins, from accumulating in the plasma. This is successfully accomplished only when the LCAT reaction balances the mechanisms that increase plasma unesterified cholesterol. A balance does not occur in familial LCAT deficiency because the enzyme is absent. It does not occur in cholesterol-fed guinea pigs because unusually large amounts of dietary unesterified cholesterol enter the plasma through inadequate control in either the intestine or the liver. It does not occur in cholestasis because phospholipid bilayers are formed and promote the accumulation of unesterified cholesterol in plasma through increased hepatic biosynthesis. Unesterified cholesterol becomes associated with the surfaces of newly formed lipoproteins by physical equilibration within the cells of the intestinal mucosa and the liver. The function of the LCAT reaction is to transport unesterified cholesterol synthesized in peripheral tissues to the liver.

The mechanism of the plasma cholesterol esterification reaction: plasma fatty acid transferase.

The mechanism of cholesterol esterification in plasma incubated at 37° has been further studied. The decrease in free cholesterol during incubation is accompanied by an approximately equimolar decrease in lecithin. The initial rate of the cholesterol esterification reaction in vitro in the plasma of normal human subjects is about 110 μmoles/l plasma/h. During incubation a large proportion of the fatty acids which become esterified to cholesterol are unsaturated, resembling in distribution the fatty acids of the C-2 position of the plasma lecithin. Esterification is reversibly inhibited by p-hydroxymercuribenzoate. Taken in conjunction with previous results, the present findings support the hypothesis that cholesterol esters are formed in plasma by the action of a fatty acid transferase, that the majority of the transesterified fatty acids originate from teh C-2 position of the plasma lecithin, and that the reaction may be of considerable importance in vivo.

SOME PROPERTIES OF A CHOLESTEROL ESTERIFYING ENZYME IN HUMAN PLASMA.

Abstract A method for the radioassay of plasma cholesterol esterifying activity has been developed. Employing this method, a study has been made of the influence of pH, polyvalent cations and anions, urea, and sodium taurocholate on the cholesterol esterification reaction in human plasma. The optimal pH range is 7.5–8.5. Calcium inhibits and phosphate stimulates the reaction, but marked effects occur only in greater than physiological concentrations. The reaction is irreversibly inactivated by urea, while the inhibition by sodium taurocholate is reversible. The behavior of the cholesterol esterifying activity of human plasma on electrophoresis, DEAE-cellulose chromatography, gel filtration, ammonium sulfate precipitation, and hydroxylapatite chromatography has also been investigated; and a procedure has been developed for the preliminary purification of a cholesterol esterifying enzyme from human blood-bank plasma.

The esterification in vitro of free cholesterol in human and rat plasma

The transformation in vitro of the plasma free cholesterol first observed by Sperry has been further studied. The drop in free cholesterol occuring on the incubation of rat plasma has been shown to be due to esterification by fatty acids. Radioactive cholesterol incubated in vitro in rat or human plasma becomes incorporated into each of the cholesterol ester subfractions obtained by silicic acid column chromatography. Under similar conditions of incubation no incorporation of free fatty acids into any of the plasma ester fractions has been demonstrated. However, when [14C]linoleic acid-labeled lecithin is incubated in vitro with rat or human plasma, radioactive cholesterol linoleate is formed as well as 14C free fatty acid, triglycerides, mono- and diglycerides, and probably also cephalin. Similarly, when [14C]palmitic acid-labeled tripalmitin is incubated with rat or human plasma, [14C]cholesterol palmitate results as well as 14C-labeled free fatty acid, mono- and diglycerides, cephalin and lecithin. It is concluded that the preformed fatty acid esters of the plasma are the source of the fatty acids for the cholesterol esterification reaction in vitro.

Fish, Fatty Acids, and Human Health

Three studies published in this issue of the Journal support the possibility that the consumption of fish may be of special benefit to human health. One of the studies1 shows that the consumption o...

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: physical and chemical studies of low and high density lipoproteins

LOW DENSITY LIPOPROTEINS (LDL) AND HIGH DENSITY LIPOPROTEINS (HDL) FROM THE PLASMA OF PATIENTS WITH FAMILIAL LECITHIN: cholesterol acyltransferase (LCAT) deficiency have been characterized by gel filtration, analytical ultracentrifugation, and gel electrophoresis, and their relative content of lipid and protein has been determined. The LDL of d 1.019-1.063 g/ml show marked heterogeneity. A subfraction of the LDL emerges from columns of 2% agarose gel with the void volume, has corrected flotation rates (S(f) degrees ) in the range of 20-400, and contains 4-10 times as much unesterified cholesterol, phosphatidylcholine, and triglyceride per mg protein as normal LDL. A major subfraction of the LDL emerges from the gel in the same general position as normal LDL, but exhibits somewhat higher flotation rates and contains 1.5-3 times as much unesterified cholesterol and phosphatidylcholine and 13 times as much triglyceride per mg protein. The HDL, shown to be heterogeneous in earlier studies, are mainly comprised of molecules which have flotation rates of F(1.20) 3-20, migrate in the alpha(1)-alpha(2) region on electrophoresis, and contain about 12 times as much unesterified cholesterol and 5 times as much phosphatidylcholine per mg protein as normal HDL. Smaller molecules are also detected, which have flotation rates of F(1.20) 0-3, migrate in the prealbumin region on electrophoresis, and contain only slightly more unesterified cholesterol and phosphatidylcholine per mg protein than normal HDL.

Further studies of the mechanism of the plasma cholesterol esterification reaction

The role of the major plasma lipoprotein fractions in the plasma cholesterol esterification reaction has been studied. Human plasma was incubated at 37° for 24 h, and the lipoproteins were subsequently fractionated by ultracentrifugal flotation. Each fraction was compared to the corresponding non-incubated control with respect to content of total and unesterified cholesterol, lecithin, and lysolecithin. Although the total cholesterol content of the individual fractions did not change as a result of the incubation, the unesterified cholesterol of each decreased, the greatest decrement being associated with the low-density lipoproteins. In contrast, the greatest decrement in lecithin was in the high-density fraction, and the greatest increment in lysolecithin was in the “very high-density” fraction. When lipoprotein fractions obtained by precipitation with ethanol were incubated separately, it was found that the relative decrease in α-lipoprotein unesterified cholesterol was much greater than that in β-lipoprotein unesterified cholesterol, and comparable results were obtained with lipoproteins prepared by differential flotation. The results suggest that the high-density lipoprotein fraction is of particular importance in vitro as a lecithin donor, and that an important factor leading to the net formation of cholesterol esters at the expense of the plasma lecithin may be the dissociation of the resulting lysolecithin from the site of transesterification.

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca2+‐triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca2+‐evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca2+‐dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense‐core secretory granules. However, replacement of the two arginines abolished the ability of the GTP‐bound form of Rab3 to inhibit exocytosis of catecholamine‐ and insulin‐secreting cells. We propose that a Rab3–calmodulin complex generated by elevated Ca2+ concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

Cloning of a Phosphatidic Acid-preferring Phospholipase A1 from Bovine Testis

We report the molecular cloning and expression of a phosphatidic acid-preferring phospholipase A1 from bovine testis. The open reading frame encoded an 875-amino acid protein with a calculated molecular mass of 97,576 daltons and a pI of 5.61. The sequence included a region similar to a lipase consensus sequence containing the putative active site serine and also included a potential, coiled-coil-forming region. Expression of the open reading frame in COS1 cells resulted in a 20–44-fold increase in phosphatidic acid phospholipase A1 activity over that of control cells. Mutation of the putative active site serine (amino acid 540) demonstrated that it was essential for this increase in enzyme activity. Northern blot analysis revealed at least five different messages with the highest overall message levels in mature testis, but detectable message in all tissues examined. Two possible alternately spliced regions in the open reading frame also were identified. Finally, a search of the data base identified six related proteins: a potential counterpart of the phospholipase A1 inCaenorhabditis elegans, two putative lipases in yeast, and three proteins separately encoded by the Drosophila retinal degeneration B gene and its mouse and human homologues.