Lipoprotein as a regulator of starch synthesizing enzyme activity

Amylose precipitating factor, a lipoprotein, functions as a regulator of in vitro activity of glycogen/starch phosphorylase and of A/UDPglucose glucosyltransferase. The results suggest that this lipoprotein could act to stimulate the in vivo production by phosphorylase of long, linear glucans (amylose) from the short chain precursors. The lipoprotein also appears to switch A/UDPglucose glucosyltransferase from the elongation of branched glucan molecules (amylopectin and glycogen) to the elongation of linear glucans (amylose).     

Changes in starch-bound lysophospholipids and lysophospholipase in germinating glacier and Hi amylose glacier barley varieties

Total starch, amylose content and amylose-included lipid phosphorus and lysophosphatidylcholine (LPC) were measured in normal Glacier (G) and Hi Amylose Glacier (HA) barley varieties during germination. From days three to six, alkaline and acidic lysophospholipase (LPL) activities in the starchy endosperm were measured and the distribution of these activities between a soluble and particulate form determined. During germination the amylose content of the starches increases as the total starch levels decline. The starch-bound LPC and lipid phosphorus disappear at the same rate between days three and six in both barley varieties, indicating no discrimination among the different lipid-included amylose population for degradation. However, both lipid phosphorus and LPC disappear more rapidly in the G than in the HA variety. This is presumably due to the slightly larger content of LPC per mg amylose of the G than of the HA variety, equivalent to 134 and 150 anhydroglucose residues per lipid molecule in G and HA, respectively. There is no increase in starch-bound lipid phosphorus or LPC expressed as nmol of phosphorus or LPC per mg amylose as amylose content declines, indicating no selective resistance of lipid-included amylose to degradation. The alkaline and acidic LPC activities in each variety increase 2–4-fold between days four and five. In both varieties ca 30% of the acidic LPL and ca 50–60% of the alkaline LPL is particulate from days three to six. No correlation can be made between the content of amylose or amylose-included lipid and particulate LPL activity. However, the possibility that particulate LPL activity is associated with specific populations of residual amylose-included lipid molecules cannot be excluded.     

An inducible humoral factor in the American cockroach (Periplaneta americana): Precipitin activity that is sensitive to a proteolytic enzyme

The humoral factor induced in the American cockroach, Periplaneta americana, by the soluble protein venoms, honeybee venom (HBT) and Western cottonmouth moccasin venom (CMV), was shown to behave like a precipitating antibody-like molecule, since it formed specific precipitin bands with homologous antigen in Ouchterlony gels. In addition, the humoral factor was demonstrated to be protein in nature, since it was sensitive to the proteolytic enzyme trypsin. Hyperimmune hemolymph treated with trypsin no longer passively protected animals from the lethal effects of HBT, and lost its ability to form precipitin bands with the antigen in Ouchterlony gels.     

A lipoproten from the membranes of Streptococcucs pyogenes and its stabilized L-form

The lack of cell wall formation by a stabilized L-form from Streptococcus pyogenes may be related to, or reflected in, changes of a particular type of membrane lipid. Therefore, this study details the first comparative investigation of isolated membranes from this Streptococcus and its stabilized L-form of isoprenoid-containing components. A lipoprotein present in minute amounts in the membranes from both this Streptococcus and its derived L-form was detected, isolated, purified and partially characterized. Lipoprotein from both membrane sources appeared to be identical, contained phosphorus and was electrophoretically homogeneous. A ratio of streptococcal to L-form membrane lipoprotein of at least 10 was observed. Chemical, physical and chromatographic studies of isolated and nonsaponifiable lipid of lipoprotein protein indicated the absence of quinones but the presence of isoprenoid units and hydroxyl group(s). Also, the spectral characteristics of lipid of lipoprotein and its chromatographic behavior, before and after acetylation, were similar to those of an isoprenoid alcohol isolated from lactobacilli and Staphylococcus aureus by others and known to be involved in bacterial cell wall peptidolgycan biosynthesis. Protein of lipoprotein, seemingly covalently linked to lipid, was unique because of its high ornithine content: with all of the ornithine of the coccal and L-form membrane apparently concentrated within this membrane component. Approximately one-half of this lipoprotein was composed of protein. The possibility of lipoprotein being related to an inability of this L-form to synthesize a rigid cell wall is indicated.     

Starch in Fungi: III. Isolation and Properties of an Amylose-precipitating Factor from Lentinellus Ursinus Fruit Bodies

A factor which precipitates amylose has been isolated from Lentinellus ursinus (Fr.) Kühner fruit bodies. This factor could be a protein or a polypeptide. Glucose, maltose, and amylopectin do not affect the binding of amylose. Amylose binding is unaffected by temperature (4 to 40 C) or pH (6 to 8.5).     

Regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase. In vitro inhibition by a protein present in bile.

The activity of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of cholesterol biosynthesis, is inhibited in vitro by factors present in both rat and bovine bile. The inhibitory factor from bovine bile has been purified to near homogeneity and is a high molecular weight lipoprotein with a density (p = 1.024) and lipid composition similar to serum β-lipoprotein. Analysis of the interaction of the enzyme and inhibitor demonstrate that the observed inactivation/inhibition is a function of lipoprotein concentration, microsomal protein concentration and duration of interaction. The observed inhibition is apparently irreversible and while neither substrate alone protects the enzyme, both substrates decrease the rate of inactivation several fold.     

Crystal structure of the koh-amylose complex

The crystal structure of potassium hydroxide complexed amylose, obtained by heterogeneous deacetylation of amylose triacetate, has been determined through a combined stereochemical structure-refinement and X-ray diffraction-analysis. The structure crystallizes in an orthorhombic unit-cell with parameters a  8.84, b  12.31, and c (fiber repeat)  22.41 Å, and with P2₁2₁2₁ symmetry. The conformation of the amylose chain is a distorted, left-handed helix with 6 d-glucose residues per turn. Each three-residue asymmetric unit is complexed with one molecule of potassium hydroxide and three molecules of water. The K⁺ ion coordinates with four oxygen atoms of the amylose chain and with two other oxygen atoms, and this coordination is probably the cause for the more-extended amylose chain-conformation than would be predicted from a φ, ψ map. The distortions in the chain are primarily manifested by different O-6 rotations and by slightly different bridge and φ, ψ angles for the individual residues. The structure is extensively hydrogen bonded, although largely through water molecules, which accounts for its ready water solubility. The left-handed conformation of the chain in this structure is consistent with the conformations of amylose triacetate and V-amylose, both of which are left-handed.     

Amylose–dye complexes in cationic micelles: an optical spectroscopy study

The formation of amylose complexes with rose bengal (RB), erythrosine B (ER), and phenolphthalein (PP) in the presence of the cationic detergent tetradecyltrimethylammonium bromide (TTABr) was studied using optical spectroscopy methods. Absorption spectroscopy, steady-state fluorescence spectroscopy and picosecond time-resolved fluorescence spectroscopy were used to derive association constants k_s of the dyes, critical micelle concentration (CMC) values and structural information on the complexes formed. It seems that PP fits very well into amylose sites, where it forms an efficient inclusion complex with k_s=44,500 M⁻¹. The molecular diameter of RB is too big to fit the amylose cavity. Only part of the xanthene unit may be adopted in the helical cavity of amylose, whereas most of the interaction occurs through electrostatic and/or dipole–dipole interactions with the amylose chain. The ER molecule is an intermediate case, because it may fit the amylose cavity or adsorb on the amylose surface to form a complex. The presence of a surfactant in the amylose–ligand system increases the association constant for all dyes. In the presence of amylose, a decrease of the detergent CMC value of about one order of magnitude is observed. It is probable that the increased number of micelles incorporate more dyes into the amylose vicinity, which finally changes the structure of the amylose chain. On a macro scale, it was noted that the samples with dyes and detergent have a lower tendency to precipitate and the gelation process is delayed compared to that in water.     

In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Locusta migratoria

Both adipokinetic hormone and octopamine have a stimulating effect on lipid release from locust fat body in vitro, when incubated in diluted haemolymph. The presence of adipokinetic hormone results in the formation of the flight-specific haemolymph lipoprotein A⁺ accepting the increased amount of lipids released into the incubation medium. In contrast, interconversions of lipoproteins do not occur when octopamine is added to the incubation medium, which is in line with the expectations: the lipid-mobilizing effect of octopamine is a limited and short-term effect. When fat body tissue is incubated with isolated haemolymph protein fractions, the lipid-mobilizing effect of adipokinetic hormone only occurs when the incubation medium contains both lipoprotein, A_y and protein fraction C, resulting in the formation of lipoprotein A⁺. In similar control incubations with the hormone omitted, some lipoprotein A⁺ is also formed (concomitant with a slight amount of lipid released), though significantly less than in incubations with hormone. Besides a stimulating function on lipolytic processes in the fat body, adipokinetic hormone is suggested to influence haemolymph lipoprotein rearrangement. A possible counteracting function of another factor in the haemolymph is discussed.     

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague–Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.     

Identification of Staphylococcus aureus Colony-Spreading Stimulatory Factors from Mammalian Serum

Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum.     

Distribution of oleoyl-estrone in rat plasma lipoproteins.

Pooled adult normal rat plasma was used for the separation of lipoprotein fractions: VLDL, LDL and HDL, from which a total lipids extract was obtained. The presence of fragments with the MW of estrone and oleoyl-estrone in the lipoprotein fractions was analyzed by HPLC-MS. The results show that oleoyl-estrone is the major estrone component in lipoproteins; this molecular species was present in all three lipoprotein lipid extracts. The lipoprotein fractions were used for the analysis of protein and lipid classes: triacylglycerols, total and esterified cholesterol and phospholipids as well as acyl-estrone. About half of the total acyl-estrone was in the HDL fraction and only about 10% in the VLDL fraction. HDLs contained about one molecule in 50 particles, LDLs one molecule per particle and VLDLs 15 molecules per particle, i.e. given their size, the larger lipoproteins contained more oleoyl-estrone than the HDLs. The distribution of this hormone suggests that oleoyl-estrone is lost with other lipids as the lipoproteins shrink. The results presented show that oleoyl-estrone is a molecule found naturally in rat lipoproteins in low concentrations - the lowest in HDLs - that are consistent with its postulated role in the control of body weight.     

Fractionation of a commercial cellulase preparation from Penicillium funiculosum and its use in the purification of a water soluble α-glucan from milled barley

A commercial enzyme preparation from a selected strain of Penicillium funiculosum has been partially purified using a single stage chromatofocusing fractionation to produce an amylase-free mixture of hydrolytic enzymes. This mixture has been used to remove the non-starch polysaccharides from aqueous extracts of laboratory milled barley. The structure of the resulting purified α-glucan fraction has been examined by gel filtration before and after enzymic debranching and by iodine staining. The mild damage caused to the starch within the barley kernel releases a low molecular weight amylopectin molecule with no detectable amylose in the product. In this respect the product is different from that produced after severe, direct grinding of the purified barley starch where some amylose appears in the water soluble extract. Although the small amount of α-glucan is not of any quantitative industrial significance in itself, it does reflect the extent and type of physical damage which is taking place in the starch granule. The model proposed to explain these results - a starch granule with a solid amylose/amylopectin core but with a number of patches of protruding amylopectin clusters - may have important implications in an industrial context.     

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

When amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose–lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose–lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose–lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose–lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.     

Apolipoprotein B-containing lipoproteins in retinal aging and age-related macular degeneration

The largest risk factor for age-related macular degeneration (ARMD) is advanced age. With aging, there is a striking accumulation of neutral lipids in Bruch''s membrane (BrM) of normal eye that continues through adulthood. This accumulation has the potential to significantly impact the physiology of the retinal pigment epithelium (RPE). It also ultimately leads to the creation of a lipid wall at the same locations where drusen and basal linear deposit, the pathognomonic extracellular, lipid-containing lesions of ARMD, subsequently form. Here, we summarize evidence obtained from light microscopy, ultrastructural studies, lipid histochemistry, assay of isolated lipoproteins, and gene expression analysis. These studies suggest that lipid deposition in BrM is at least partially due to accumulation of esterified cholesterol-rich, apolipoprotein B-containing lipoprotein particles produced by the RPE. Furthermore, we suggest that the formation of ARMD lesions and their aftermath may be a pathological response to the retention of a sub-endothelial apolipoprotein B lipoprotein, similar to a widely accepted model of atherosclerotic coronary artery disease (Tabas, I., K. J. Williams, and J. Borén. 2007. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation. 116:1832–1844). This view provides a conceptual basis for the development of novel treatments that may benefit ARMD patients in the future.     

Structural characterization of the inflammatory moiety of a variable major lipoprotein of Borrelia recurrentis

Louse-borne relapsing fever, caused by Borrelia recurrentis, provides one of the best documented examples of the causative role of tumor necrosis factor (TNF) in the pathology of severe infection in humans. We have identified the principal TNF-inducing factor of B. recurrentis as a variable major lipoprotein (Vmp). Here we report the complete gene sequence of Vmp, including its lipoprotein leader sequence. Using metabolically labeled forms of the native Vmp we confirm that the TNF inducing properties are associated with the lipid portion of the molecule. Quadrupole orthogonal time of flight mass spectrometry unequivocally locates the lipidic moiety at the NH(2)-terminal cysteine of the native polypeptide, and indicates the existence of three forms which are consistent with the structures C16:0, C16:0, C16:0 glyceryl cysteine; C18:1, C16:0, C16:0 glyceryl cysteine; and C18:0, C16:0, C16:0 glyceryl cysteine. These data provide the first direct evidence that the TNF inducing lipid modification of native Borrelia lipoproteins is a structural homologue of the murein lipoprotein of Escherichia coli.     

Lipid exchange and transfer between biological lipid-protein structures

The dynamic state of membrane and lipoprotein lipids is all the more impressive when the complexity of lipoprotein and membrane structure is considered. For as long as such a ubiquitous and easily demonstrable process has been studied, the mechanism(s) of lipid exchange is still unknown. Is a direct contact between lipoproteins and membranes required for lipid exchange, or are molecules expelled from lipid-protein complexes to spend a transient existence in the aqueous environment before returning to their donor or being accomodated in another complex? Although recent studies suggest the certain proteins such as the phospholipid exchange proteins can exert some vectoral and selective control over exchange reactions, the exchange of lipids, as studied under most conditionsin vitro, seems to be a random occurrence and a purely physicochemical event. Ifin vitro studies are indeed reflective of the processesin vivo, the lipid exchange activity in a cell can likely be depicted as shown in Figure 18.     

Double Superhelix Model of High Density Lipoprotein

High density lipoprotein (HDL), the carrier of so-called “good” cholesterol, serves as the major athero-protective lipoprotein and has emerged as a key therapeutic target for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation and selective isotopic deuteration to the study of nascent HDL to obtain the low resolution structure in solution of the overall time-averaged conformation of apolipoprotein AI (apoA-I) versus the lipid (acyl chain) core of the particle. Remarkably, apoA-I is observed to possess an open helical shape that wraps around a central ellipsoidal lipid phase. Using the low resolution SANS shapes of the protein and lipid core as scaffolding, an all-atom computational model for the protein and lipid components of nascent HDL was developed by integrating complementary structural data from hydrogen/deuterium exchange mass spectrometry and previously published constraints from multiple biophysical techniques. Both SANS data and the new computational model, the double superhelix model, suggest an unexpected structural arrangement of protein and lipids of nascent HDL, an anti-parallel double superhelix wrapped around an ellipsoidal lipid phase. The protein and lipid organization in nascent HDL envisages a potential generalized mechanism for lipoprotein biogenesis and remodeling, biological processes critical to sterol and lipid transport, organismal energy metabolism, and innate immunity.     

A V7 conformation of dimethyl sulfoxide-amylose complex

X-ray investigation of a crystalline complex between dimethyl sulfoxide (DMSO) and amylose indicates a sevenfold helical structure having an orthogonal unit cell: a = 30.23 Å, b = 28.18 Å, and C = 7.91 Å (helix axis). This helical amylose results from precipitation of amylose in DMSO solutions under suitably dry conditions using toluene as a precipitating agent. Analyses show a molar ratio of 0.95:2:0.4:0.08 for DMSO, amylose glucose units, water, and toluene, respectively. DMSO and amylose combine in at least two crystalline conformations, sixfold and sevenfold helices. The amount of water present influences the resulting conformation. The existence of the sevenfold helical solid indicates that the amylose sevenfold helix has greater stability than previously recognized by conformational energy calculations. In general, potential energy calculations cannot be correct that ignore the influence of guest molecules and their effects upon the maltose residue conformation and that lead to a sixfold helix as the most probable structure.     

Structural models of human apolipoprotein A-I: a critical analysis and review

Human apolipoprotein (apo) A-I has been the subject of intense investigation because of its well-documented anti-atherogenic properties. About 70% of the protein found in high density lipoprotein complexes is apo A-I, a molecule that contains a series of highly homologous amphipathic alpha-helices. A number of significant experimental observations have allowed increasing sophisticated structural models for both the lipid-bound and the lipid-free forms of the apo A-I molecule to be tested critically. It seems clear, for example, that interactions between amphipathic domains in apo A-I may be crucial to understanding the dynamic nature of the molecule and the pathways by which the lipid-free molecule binds to lipid, both in a discoidal and a spherical particle. The state of the art of these structural studies is discussed and placed in context with current models and concepts of the physiological role of apo A-I and high-density lipoprotein in atherosclerosis and lipid metabolism.