Differential Stability of High-density Lipoprotein Subclasses: Effects of Particle Size and Protein Composition

High-density lipoproteins (HDLs) are complexes of proteins (mainly apoA-I and apoA-II) and lipids that remove cholesterol and prevent atherosclerosis. Understanding the distinct properties of the heterogeneous HDL population may aid the development of new diagnostic tools and therapies for atherosclerosis. Mature human HDLs form two major subclasses differing in particle diameter and metabolic properties, HDL₂ (large) and HDL₃ (small). These subclasses are comprised of HDL(A-I) containing only apoA-I, and HDL(A-I/A-II) containing apoA-I and apoA-II. ApoA-I is strongly cardioprotective, but the function of the smaller, more hydrophobic apoA-II is unclear. ApoA-II is thought to counteract the cardioprotective action of apoA-I by stabilizing HDL particles and inhibiting their remodeling. To test this notion, we performed the first kinetic stability study of human HDL subclasses. The results revealed that the stability of plasma spherical HDL decreases with increasing particle diameter; which may facilitate preferential cholesterol ester uptake from large lipid-loaded HDL₂. Surprisingly, size-matched plasma HDL(A-I/A-II) showed comparable or slightly lower stability than HDL(A-I); this is consistent with the destabilization of model discoidal HDL observed upon increasing the A-II to A-I ratio. These results clarify the roles of the particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of apoA-II in this remodeling.     

Degradation of serum amyloid A and apolipoproteins by serum proteases

We have investigated the protease activity, present in human serum, that digests the serum amyloid A (SAA) protein. SAA radiolabeled with 125I was incubated at 37 degrees C with serum and plasma and analyzed for degradation products by alkaline urea-polyacrylamide gel electrophoresis and gel filtration chromatography. Serum initially digested SAA to intermediates of 3000-5000 in molecular weight, and these were further degraded to smaller peptides with prolonged incubation. SAA was not degraded by plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA) or heparin. Recalcification of plasma anticoagulated with EDTA led to the generation of protease activity against SAA whereas EDTA plasma defibrinated with thrombin was inactive. We employed both nonselective and selective protease inhibitors and synthetic substrates for kallikrein and plasmin to further characterize the serum protease. These studies demonstrated that degradation of SAA is not directly attributable to enzymes involved in coagulation, kinin formation, or fibrinolysis, but the unidentified protease may be activated by one of the clotting factors. The specificity of the SAA degradation was demonstrated in experiments with three of the well-characterized apolipoproteins. Apolipoproteins A-I, C-I, and C-III-1, which also associate with the plasma high-density lipoproteins, were not degraded by serum although they were good substrates for purified thrombin and plasmin.     

Expression of serum amyloid A protein in the absence of the acute phase response does not reduce HDL cholesterol or apoA-I levels in human apoA-I transgenic mice

Plasma concentrations of high density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo)A-I are significantly decreased in inflammatory states. Plasma levels of the serum amyloid A (SAA) protein increase markedly during the acute phase response and are elevated in many chronic inflammatory states. Because SAA is associated with HDL and has been shown to be capable of displacing apoA-I from HDL in vitro, it is believed that expression of SAA is the primary cause of the reduced HDL cholesterol and apoA-I in inflammatory states. In order to directly test this hypothesis, we constructed recombinant adenoviruses expressing the murine SAA and human SAA1 genes (the major acute phase SAA proteins in both species). These recombinant adenoviruses were injected intravenously into wild-type and human apoA-I transgenic mice and the effects of SAA expression on HDL cholesterol and apoA-I were compared with mice injected with a control adenovirus. Plasma levels of SAA were comparable to those seen in the acute phase response in mice and humans. However, despite high plasma levels of murine or human SAA, no significant changes in HDL cholesterol or apoA-I levels were observed. SAA was found associated with HDL but did not specifically alter the cholesterol or human apoA-I distribution among lipoproteins. In summary, high plasma levels of SAA in the absence of a generalized acute phase response did not result in reduction of HDL cholesterol or apoA-I in mice, suggesting that there are components of the acute phase response other than SAA expression that may directly influence HDL metabolism.     

Biogenesis of HDL by SAA is dependent on ABCA1 in the liver in vivo

Serum amyloid A (SAA) was markedly increased in the plasma and in the liver upon acute inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) in mice, and SAA in the plasma was exclusively associated with HDL. In contrast, no HDL was present in the plasma and only a small amount of SAA was found in the VLDL/LDL fraction (d < 1.063 g/ml) after the induction of inflammation in ABCA1-knockout (KO) mice, although SAA increased in the liver. Primary hepatocytes isolated from LPS-treated wild-type (WT) and ABCA1-KO mice both secreted SAA into the medium. SAA secreted from WT hepatocytes was associated with HDL, whereas SAA from ABCA1-KO hepatocytes was recovered in the fraction that was >1.21 g/ml. The behavior of apolipoprotein A-I (apoA-I) was the same as that of SAA in HDL biogenesis by WT and ABCA1-KO mouse hepatocytes. Lipid-free SAA and apoA-I both stabilized ABCA1 and caused cellular lipid release in WT mouse-derived fibroblasts, but not in ABCA1-KO mouse-derived fibroblasts, in vitro when added exogenously. We conclude that both SAA and apoA-I generate HDL largely in hepatocytes only in the presence of ABCA1, likely being secreted in a lipid-free form to interact with cellular ABCA1. In the absence of ABCA1, nonlipidated SAA is seemingly removed rapidly from the extracellular space.     

Serum amyloid A protein generates pre beta 1 high-density lipoprotein from alpha-migrating high-density lipoprotein

Serum amyloid A protein (SAA), an acute-phase reactant in reactive amyloidosis, has high affinity for high-density lipoprotein (HDL). When SAA is added to HDL, SAA displaces apolipoprotein A-I (apoA-I) and phospholipid from the HDL particles. These dissociated components may form prebeta1-HDL because free apoA-I can associate with phospholipid to become a lipoprotein having prebeta mobility. To determine whether SAA generates prebeta1-HDL from alpha-migrating HDL, we investigated the effects of recombinant SAA on HDL subfraction concentration using nondenaturing two-dimensional gradient gel electrophoresis. When we added SAA (0.5 mg/mL) to plasma, the prebeta1-HDL concentration increased by 164% (from 4.7% +/- 1.3% to 12.4% +/- 3.2% of apoA-I, p < 0.005). The increase in prebeta1-HDL was proportional to the dose of SAA. When we added SAA to a column of Sepharose beads coupled to the isolated HDL (alpha-migrating HDL), prebeta1-HDL was dissociated from the column together with the SAA-associated HDL. In summary, we demonstrate that SAA generates prebeta1-HDL from alpha-migrating HDL. We speculate that SAA-mediated HDL remodeling may take place in inflammation.     

Impact of serum amyloid A on high density lipoprotein composition and levels

Serum amyloid A (SAA) is an acute-phase protein mainly associated with HDL. To study the role of SAA in mediating changes in HDL composition and metabolism during inflammation, we generated mice in which the two major acute-phase SAA isoforms, SAA1.1 and SAA2.1, were deleted [SAA knockout (SAAKO) mice], and induced an acute phase to compare lipid and apolipoprotein parameters between wild-type (WT) and SAAKO mice. Our data indicate that SAA does not affect apolipoprotein A-I (apoA-I) levels or clearance under steady-state conditions. HDL and plasma triglyceride levels following lipopolysaccharide administration, as well as the decline in liver expression of apoA-I and apoA-II, did not differ between both groups of mice. The expected size increase of WT acute-phase HDL was surprisingly also seen in SAAKO acute-phase HDL despite the absence of SAA. HDLs from both mice showed increased phospholipid and unesterified cholesterol content during the acute phase. We therefore conclude that in the mouse, SAA does not impact HDL levels, apoA-I clearance, or HDL size during the acute phase and that the increased size of acute-phase HDL in mice is associated with an increased content of surface lipids, particularly phospholipids, and not surface proteins. These data need to be transferred to humans with caution due to differences in apoA-I structure and remodeling functions.     

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

Influence of apoA-I and apoE on the formation of serum amyloid A-containing lipoproteins in vivo and in vitro

Serum amyloid A (SAA) circulates bound to HDL3 during the acute-phase response (APR), and recent evidence suggests that elevated levels of SAA may be a risk factor for cardiovascular disease. In this study, SAA-HDL was produced in vivo during the APR and without the APR by injection of an adenoviral vector expressing human SAA-1. SAA-HDL was also produced in vitro by incubating mouse HDL with recombinant mouse SAA and by SAA-expressing cultured hepatoma cells. Whether produced in vivo or in vitro, SAA-HDL floated at a density corresponding to that of human HDL3 (d 1.12 g/ml) separate from other apolipoproteins, including apolipoprotein A-I (apoA-I; d 1.10 g/ml) when either apoA-I or apolipoprotein E (apoE) was present. In the absence of both apoA-I and apoE, SAA was found in VLDL and LDL, with low levels in the HDL and the lipid-poor fractions suggesting that other HDL apolipoproteins are incapable of facilitating the formation of SAA-HDL. We conclude that SAA does not exist in plasma as a lipid-free protein. In the presence of HDL-associated apoA-I or apoE, SAA circulates as SAA-HDL with a density corresponding to that of human HDL3. In the absence of both apoA-I and apoE, SAA-HDL is not formed and SAA associates with any available lipoprotein.     

Uptake of endogenous cholesterol by a synthetic lipoprotein

The addition of cholesterol-poor phospholipid liposomes to canine plasma in vivo and in vitro substantially alters the distribution of phospholipids, apoproteins, and, especially, cholesterol. In vivo, intravenously injected phospholipid liposomes remain discrete particles, which are readily distinguished from the normally occurring lipoproteins by their buoyant density and electrophoretic mobility. They acquire unesterified cholesterol from endogenous sources, thereby producing an acute rise in the concentration of this sterol in plasma. The liposomes also accumulate endogenous proteins, one of which is identified as apolipoprotein A-I. In vitro, phospholipid liposomes incubated with plasma acquire unesterified cholesterol and apolipoprotein A-I at the expense of high-density lipoproteins (HDL), the major carrier of cholesterol in normal canine plasma. In exchange, the HDL particles are enriched in phospholipids and become larger. At sufficiently high concentrations, the liposomes nearly completely deplete HDL of its unesterified cholesterol. Thus, there are generated two types of particles, both rich in apolipoprotein A-I and phospholipid, but one (modified HDL) containing mainly esterified cholesterol in its core and the other (modified liposomes) containing mainly unesterified cholesterol at its surface. It is concluded that phospholipid liposomes produce important changes in the distribution of lipids and protein in canine plasma, particularly at the expense of HDL. These changes appear to favor the mobilization of tissue cholesterol into the plasma, and may have application to atherosclerosis.     

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes

Ecto-F₁-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F₁-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y₁₃-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F₁-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F₁-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F₁-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes.This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.     

Polymorphisms in the SAA1/2 gene are associated with carotid intima media thickness in healthy Han Chinese subjects: the Cardiovascular Risk Survey

Background

Serum amyloid A protein (SAA) is not only an inflammatory factor, but also an apolipoprotein that can replace apolipoprotein A1 (apoA1) as the major apolipoprotein of high-density lipoprotein (HDL), which has been linked to atherosclerosis. However, the relationship between genetic polymorphisms of SAA and the intima-media thickness (IMT) of the common carotid artery in healthy subjects remains unclear. We investigated the role of SAA1 and SAA2 gene polymorphisms with IMT in a cohort of healthy subjects participating in the Cardiovascular Risk Survey (CRS) study.

Methodology/Principal Findings

Anthropometric and B-mode ultrasound of the carotid IMT were measured in 1914 subjects (849 men; 1065 women) recruited from seven cities in Xinjiang province, (western China). Four SNPs (rs12218, rs2229338, rs1059559, and rs2468844) were genotyped by use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The SNP rs12218 was associated with carotid IMT by analyses of a dominate model (P<0.001) and additive model (P = 0.003), and the difference remained significant after multivariate adjustment (P = 0.008, P<0.001, respectively). This relationship was also observed in rs2468844 after multivariate adjustment by recessive model analysis (P = 0.011) but this was not observed in rs2229338 and rs1059559 before and after multivariate adjustment. These associations were not modified by serum HDL concentration. Furthermore, there were significant interactions between rs2468844 and rs12218 (interaction P<0.001) and rs2229338 (interaction P = 0.001) on carotid IMT.

Conclusion/Significance

Both rs12218 of the SAA1 gene and rs2468844 of SAA2 gene are associated with carotid IMT in healthy Han Chinese subjects.     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

A new method for the fractionation of human plasma high density lipoprotein.

We have devised a new method for the fractionation of human plasma high density lipoprotein (HDL). The HDL was chromatographed on DEAE-agarose columns using a continuous gradient of 0.06--0.15 M NaCl. The elution pattern obtained showed three phases, each with differing peptide composition. Examination of the three subfraction showed that each contained both apoA-I and apo A-II, but in different proportions. Subfraction 1 contained no apo C-II or C-III-1 and only a trace of apo C-III-2, subfraction 2 contained apo C-II and C-III-1 but no C-III-2, while subfraction 3 contained considerable apo C-III-2 with only traces of apo C-II or C-III-1.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.     

The interaction between apolipoprotein serum amyloid A and high-density lipoprotein

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins (HDLs) via its N-terminus. The murine isoform SAA2.2 forms a hexamer in solution and the N-terminus is shielded from the solvent. Therefore, it is unclear how the SAA2.2 hexamer might bind HDL. In this study, the binding of SAA2.2 to murine HDL was investigated by glutaraldehyde cross-linking and polyacrylamide gel electrophoresis. The hexamer did not bind HDL significantly at 20 degrees C. However, at temperatures between 25-30 degrees C, SAA2.2 became destabilized and its monomeric form bound to HDL. SAA2.2 binding did not significantly replace Apo A-I in HDL particles. At 37-45 degrees C SAA2.2 binds less to HDL, suggesting that its binding is weak and sensitive to physiological and pathological temperatures, and thereby, potentially modulated, in vivo, by other factors.     

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10 nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37 °C, but were almost completely denatured around 60 °C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.     

Effects of the acute phase response on the concentration and density distribution of plasma lipids and apolipoproteins

Longitudinal studies were carried out in the rabbit model to determine alterations in the concentration and density distribution of plasma lipids and apolipoproteins during the acute phase response (APR) characterized by elevated levels of C-reactive protein (CRP) and serum amyloid A (SAA). Twelve hr after the intramuscular injection of croton oil, SAA was detectable in high density lipoprotein (HDL). At the height of the response (72 hr), HDL decreased while SAA became the major HDL apoprotein, up to 80% of the proteins in the higher density fractions. The SAA-enriched particles became denser (density of HDL3) but larger (size of HDL2), had slower electrophoretic mobility, and were depleted in apoA-I, cholesterol, triglyceride, and phospholipid. HDL-cholesterol decreased and was redistributed to other fractions while apoA-I disappeared from the circulation. During this time plasma triglycerides increased 6- to 10-fold while plasma cholesterol and phospholipids showed minimal changes. ApoB increased 5- to 6-fold while the apoB-containing particles shifted to higher density resulting in elevated IDL and then LDL during recovery. VLDL (d less than 1.006 g/ml) increased and acquired 30-40% of the plasma triglycerides, cholesterol, phospholipid, and apoB. SAA also increased in VLDL while apoE decreased.     

Species-specific proteins of the 50S subunit of the chloroplast ribosome in the genus Nicotiana

Summary The large subunits (50S) of chloroplast ribosomes were isolated from Nicotiana tabacum, a species of the Western Hemisphere, and from N. excelsior and N. gossei, Australian species. Their proteins were compared by two-dimensional gel electrophoresis. A pair of proteins (T₁₂ and T₁₂) observed in N. tabacum has electrophoretic mobilities which differ from those of a similarly migrating, and probably homologous, pair of proteins observed in N. excelsior and N. gossei. The species-specific proteins in N. tabacum differ slightly in electrophoretic mobilities based on both charge and molecular weight from those in N. excelsior and N. gossei. Tryptic digests of radioiodinated proteins reveal that the peptide maps of all six proteins are similar. These results suggest that chemically altered forms of one or more proteins of the 50S chloroplast ribosome subunit may exist in vivo.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

The ribosomal proteins of Drosophila melanogaster. I. Characterization in polyacrylamide gel of proteins from larval, adult, and ammonium chloride-treated ribosomes

Summary Ribosomes were isolated from larvae and adult flies, and the purity of the preparation was checked by electron microscopy. The ribosomal proteins were extracted with cold dilute hydrochloric acid, and precipitated with cold acetone. The proteins were characterized by polyacrylamide gel electrophoresis. At pH 3.0 at least 25 bands of different color intensities were resolved, forming a complex pattern.On the basis of electrophoretic mobilities, it was shown that some ribosomal proteins are species-specific, and that larval ribosomes have three protein components more than ribosomes from adult flies.Incubation of the ribosomes with 0.75 M NH₄Cl at a low Mg⁺⁺ concentration lead to detachment of 64% of the ribosomal protein. This detachment of protein molecules was considerably reduced by a five-fold increase of Mg⁺⁺ ions.