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Semliki Forest Virus-based transient expression system was utilized to investigate the interaction of three nascent apolipoprotein E isoforms with amyloid beta peptide (Abeta) using expressed core DNA in baby hamster kidneys (BHK-21) cells. Data indicated the presence of structural features such as carbohydrate chains containing sialic acid determined the reactivity of apoE forms toward Abeta. A decrease in reaction temperature afforded equal binding efficiency for all the three isoforms.

The binding of recombinant apolipoprotein (apo(a)) with high affinity to plasminogen in solution was evaluated through binding studies using various truncated and mutant forms of r-apo(a). Results obtained revealed that the interaction was mediated through lysine binding sites present in the protease domain of apo(a). Rather than competing with plasminogen for binding to plasmin-modified fibrinogen, apo(a) formed a complex with plasminogen.

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

Fluorescence spectroscopy was utilized to determine the effects of human apolipoprotein E (apoE) on the formation of the beta-amyloid fibril (beta-Af) in vitro. Administration of apoE in freshly prepared betaAfs induced inhibited fibril formation in a dose-dependent manner. The differential effects of ApoE on beta-Af formation were also characterized by the inhibition of the nucleation phase via a mechanism that was similar to the attenuation of the elongation phase by the cholesterol transport protein.

Macrophage-derived foam cells express apolipoprotein E (apoE) abundantly in atherosclerotic lesions. To examine the physiologic role of apoE secretion by the macrophage in atherogenesis, bone marrow transplantation was used to reconstitute C57BL/6 mice with macrophages that were either null or wild type for the apoE gene. After 13 weeks on an atherogenic diet, C57BL/6 mice reconstituted with apoE null marrow developed 10-fold more atherosclerosis than controls in the absence of significant differences in serum cholesterol levels or lipoprotein profiles. ApoE expression was absent in the macrophage-derived foam cells of C57BL/6 mice reconstituted with apoE null marrow. Thus, lack of apoE expression by the macrophage promotes foam cell formation. These data support a protective role for apoE expression by the macrophage in early atherogenesis.

Hepatocyte nuclear factor 4 (HNF-4) in rats binds to site A from apolipoprotein AI (Apo AI) DNA, suppressing site A activity. Specifically, HNF-4 interferes with the enhancer role of the T3 receptor, adversely affecting the net expression of the Apo AI gene. This was gleaned from an examination of the transcription factors that influence the expression of the Apo AI gene. Results of previous studies which show that HNF-4 enhances the activity of a cis-acting site C in the rat Apo AI promoter and whether it binds to and modulates the transcriptional activity of site A, since sites C and A share 80% homology were validated.

The N-terminal 43 residues of human apolipoprotein A-I (apo hA-I) are necessary to maintain and stabilize the structure of these amino acids. They also play a crucial role in the conformational switch between the lipid-free and lipid-bound forms. Denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements and far- and near-ultraviolet circular dichroic spectroscopy showed significant structural differences between lipid-free apo (delta)(1-43)A-I and apo hA-I. According to spectral and physical measurements, apo (delta)(1-43)A-I has a folded, tertiary structure, although it is substantially less stable than that of apo hA-I.

Apolipoprotein AI (apoAI) is the principal protein constituent of highdensity lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apoAI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI [Delta]44-126, apoAI [Delta]139-170, and apoAI [Delta]190-243). Circular dichroism spectroscopy showed that the [Alpha]-helical content of lipid-free apoAI [Delta]44-126 was 27% while the other mutants and native apoAI averaged 55 [+ or -] 2%, suggesting that the missing N-terminal portion contains most of the [Alpha]-helical structure of lipid-free apoAI. ApoAI [Delta]44-126 exhibited the largest increase in [Alpha]-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for [Alpha]-helix stability in the lipid-free state, whereas the C terminus is required for [Alpha]-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the [Alpha]-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in [Alpha]-helical content observed when native apoAI binds lipid results from the formation of [Alpha]-helix primarily in the C-terminal half of the molecule.

The role of the ubiquitin-proteosome pathway in the degradation of apolipoprotein B (apoB) was analyzed in cultured human hepatoma (HepG2) cells. The adenosine phosphate-dependent ubiquitin-proteosome pathway degrades apoB by inhibiting the proteosomes of HepG2 cells. Furthermore, the role of ubiquitin-proteosome pathway on the degradation of apoB was detected by immunoprecipitation and Western blotting of radiolabeled proteins in HepG2 cells.

Male infertility, affecting as many as 10% of the adult population, is an extremely prevalent disorder. In most cases, the cause of the condition is unknown, and genetic factors that might affect male fertility, other than some sequences on the Y chromosome, have not been identified. We report here that male mice heterozygous for a targeted mutation of the apolipoprotein B (apo B) gene exhibit severely compromised fertility. Sperm from these mice failed to fertilize eggs both in vivo and in vitro. However, these sperm were able to fertilize eggs once the zona pellucida was removed but displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization-related and other experiments revealed reduced sperm motility, survival time, and sperm count also contributed to the infertility phenotype. Recognition of the infertility phenotype led to the identification of apo B mRNA in the testes and epididymides of normal mice, and these transcripts were substantially reduced in the affected animals. Moreover, when the genomic sequence encoding human apo B was introduced into these animals, normal fertility was restored. These findings suggest that this genetic locus may have an important impact on male fertility and identify a previously unrecognized function for apo B.

We have studied the effects of endogenous and exogenous estrogen on atherosclerotic lesions in apolipoprotein E-deficient mice. Female mice ovariectomized (OVX) at weaning displayed increases (P < 0.01) in fatty streak lesions in the proximal aorta and aortic sinus compared with female mice with intact ovarian function. These differences between the OVX and sham controls were apparent in both chow- and 'Western-type' diet-fed mice. Moreover, increases in lesion size following OVX occurred without changes in plasma cholesterol. Hormone replacement with subdermal 17-[Beta]-estradiol pellets releasing either 6, 14, or 28 [[micro]gram]/day significantly decreased (P < 0.001) atherosclerotic lesion area in both male and OVX female mice. In contrast, neither 17-[Alpha]-estradiol (28 [[micro]gram]/day) or tamoxifen (85 [[micro]gram]/day) affected lesion progression in OVX female mice. In the Western diet-fed group, exogenous estradiol markedly reduced plasma cholesterol and triglycerides, whereas, in animals fed the chow diet, exogenous estrogen and tamoxifen treatment only decreased plasma and very low density lipoprotein triglycerides. However, lesion area was only weakly correlated with plasma cholesterol and triglycerides, 0.35 and 0.44 tau values, respectively (P [less than] 0.01). In summary, in the apolipoprotein E-deficient mouse 17-[Beta]-estradiol protects against atherosclerotic lesion formation, and this can only be partially explained through effects on plasma lipoprotein levels.

The preparation and characterization of full-length and truncated proapolipoprotein AI (proapoAI) derived from human plasma were carried out. The protein, which is found in high-density lipoproteins, was produced using Escherichia coli/pGex vector expression. Their structural-activity relationships were examined via circular dichroism, iodide quenching and fluorescence anisotropy.

Class A exchangeable apolipoproteins and noexchangeable apolipoproteins play vital roles in the transportation of lipid-soluble molecules during lipid metabolism. The A-II, C-I, C-II and C-III apolipoproteins have the highest lipid affinity among exchangeable apolipoproteins due to the presence of amphipathic helices. Class Y helices also exhibit higher lipid affinity compared to class A1 helices. Interfacial lysine residues increase lipid affinity and assist in the anchoring of the amphipathic helix to the lipid bilayer.

The characterization of the source of the odor in the human axillary region is not only of commercial interest but is also important biologically because axillary extracts can alter the length and timing of the female menstrual cycle. In males, the most abundant odor component is known to be E-3-methyl-2-hexenoic acid (E-3M2H), which is liberated from nonodorous apocrine secretions by axillary microorganisms. Recently, it was found that in the apocrine gland secretions, 3M2H is carried to the skin surface bound to two proteins, apocrine secretion odor-binding proteins 1 and 2 (ASOB1 and ASOB2) with apparent molecular masses of 45 kda and 26 kda, respectively. To better understand the formation of axillary odors and the structural relationship between 3M2H and its carrier protein, the amino acid sequence and glycosylation pattern of ASOB2 were determined by mass spectrometry. The ASOB2 protein was identified as apolipoprotein D (apoD), a known member of the [[Alpha].sub.2[Mu]]-microglobulin superfamily of carrier proteins also known as lipocalins. The pattern of glycosylation for axillary apoD differs from that reported for plasma apoD, suggesting different sites of expression for the two glycoproteins. In situ hybridization of an oligonucleotide probe against apoD mRNA with axillary tissue demonstrates that the message for synthesis of this protein is specific to the apocrine glands. These results suggest a remarkable similarity between human axillary secretions and nonhuman mammalian odor sources, where lipocalins have been shown to carry the odoriferous signals used in pheromonal communication.

The human placental tissue expresses a 22.7 kDa protein that resembles apolipoprotein A-I (apo A-I). The presence of a single apo A-I gene in the human genome indicates that the protein results from an alternative splice of the apo A-I primary transcript. HPLC of partly purified placental extract separates two fractions that react with apo A-I antiserum. Mass spectral analysis reveals that the mass of the fractions is 22,721 daltons. The N-terminal amino acid sequences of the fractions are similar. Northern blot analysis of placental RNA detects only one band smaller than apo A-I mRNA.

A multicompartmental model of stable isotope methodology is developed to examine apolipoprotein B kinetics in very low density lipoprotein subfractions, intermediate density lipoprotein, and low density lipoprotein. The methodology is independent of dose and administration protocol. It provides estimates for the rates of production, interconversion, and catabolism of individual lipoproteins. Values for apolipoprotein B input agree with the results obtained by radioiodinated tracer techniques.

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH [approximately equal to]7, by using differential scanning calorimetry and circular dichroism. At protein concentrations

Human brain and human placental cDNA libraries were screened using the yeast two-hybrid method to search for proteins involved in the transmission of signals from activated type I (RI) and type II receptors (RII) of transforming growth factor-beta (TGF-B). The results show that a protein identical to apolipoprotein (apoJ)/clusterin interacts with both RI and RII via its C-terminal 127 amino acid chain. It was also discovered that the 60-kilodalton intracellular form of apoJ is involved in TGF-B receptor processing or TGF-B signaling.

The role of the protein moiety of lipoproteins in the action of high-density lipoprotein (HDL) as the primary acceptor of cellular cholesterol during intracellular cholesterol efflux was investigated using monoclonal antibodies against apolipoprotein AI (apoAI). The results show that a region near residues 140-150 is involved in the promotion of intracellular cholesterol efflux by apoAI.

Interleukin-6 (IL-6), IL-1-beta, and tumor necrosis factor-alpha produced by Caco-2 cells decrease the basolateral secretion of apolipoprotein B (apo B). IL-6 is the most potent inhibitor, and also blocks triacylglycerol secretion. Transforming growth factor-beta-1 (TGF-beta-1) enhances the release of apo B and triacylglycerol. TGF-beta-1 increase the synthesis and secretion of apo B, while IL-6 decreases synthesis and secretion. Decomposition of apo B is unaffected by both IL-6 and TGF-beta-1.

The application of parent human and rhesus monkey lipoprotein(a) (Lp-a-) to mild reductive conditions lead to free forms of wild type and mutant apolipoprotein(a) (apo-a-) phenotypes. The free form of apo(a)s are all positive lysine binding. The Lp(a) complexes obtained from incubation between free apo(a)s and autologous human or rhesus low-density lipoproteins, and native Lp(a) are structurally and functionally identical. The low plasma level of Lp(a) in the human mutant is related to the defective production and/or secretion of apo(a).

A study analyzes the substitutions in the protease domain of apolipoprotein(a) (apo(a)) and the replacement of Arg with Ser inactivate apo(a). The study uses a mutant with a substitution in the Arg residue containing a Ser residue to observe the proteolytic activity of a recombinant form of apo(a). Cleavage of the mutant with tissue-type plasminogen activator fails to affect the activity. The presence of a number of kringle IV repeats seems to have no effect on apo(a) and the protease region remains unmodified.

A study of the three deletion mutants apo A-I(delta-Leu44-Leu126), apo A-I(delta-Glu139-Leu170) and apo A-I(delta-Ala190-Gln243) and recombinant human apolipoprotein reveals that any mutation causing rupture of geometry in apo A-I decreases the activity of lecithin-cholesterol acyltransferase. When apo A-I(delta-Ala190-Gln243) and dimyristoylphosphatidylcholine are combined, the turbidity rate decreases 10 times as compared to other apo A-I proteins. Removal of carboxy terminal decreases the rate but not the degree of lipid binding while, removal of the amino group has little impact on lipid binding.

Amino acids 141-149 in apolipoprotein E (apoE) present in an amphipathic alpha-helix conformation comprise an immuno-regulatory sequence having either cytostatic or cytotoxic activity. The activity is determined by nonpolar face hydrophobicity and polar face charge density. By mediating removal of cholesterol-rich lipoproteins from the blood, apoE plays a crucial role in the control of cholesterol homeostasis. ApoE is an active suppressor of interlukin 2-dependent T lymphocyte proliferation.

The growing trend towards a genetic link in Alzheimer's disease also creates legal and ethical concerns. Chromosome 19 may carry a type of apolipoprotein E, a blood protein, in some patients with this progressive brain disorder. Only half of all Alzheimer's patients have the apolipoprotein E genetic configuration, rendering general screening fairly useless. Commercial DNA testing has become available but may be of limited benefit. Since no known cure exists for Alzheimer's disease, knowing in advance may cause more stress than assurance. Access to a person's genetic information needs to be protected with respect to outsiders. Future policy must address issues of confidentiality as it relates to researchers, employers, and health insurers. For example, researchers may preserve patient anonymity by using unidentifiable samples for repeated medical research purposes. Disclosure of data use and informed consent are other ethical alternatives.

Recombinant apolipoprotein(a), contained in lipoprotein(a), influences in vitro clot lysis with a dose-dependent antifibrinolytic effect involving the attenuation of tissue plasminogen activator-mediated plasminogen activation and inhibition fibrin plasmin degradation. This effect was studied using a 17-kringle form of recombinant apolipoprotein(a) on in vitro fibrin clot lysis induced by low and high thrombin concentrations. The antifibrinolytic effect may be the mechanism through which lipoprotein(a) helps vascular disease development.

A computerized image analysis was used to study A-beta (Ab) deposits of senile plaques (SP) in Alzheimer disease (AD) to determine how the genetic abnormalities of Down syndrome (DS) and apolipoprotein E epsilon4 allele (apoE e4) relate to pathological changes in the brain. Results concluded that the size distribution curve of SP is log-normal, increased Ab levels underlie trisomy 21 deposition, and increased SP initiation causes apoE e4 association with increased Ab deposition.

An isoform of the apoE family of proteins, apoE3, inhibits amyloid nucleation even at low concentrations of 40 nM. The N-terminal 191 amino acids of a proteolytic fragment of apoE3 causes this inhibitory activity, which involves suppression of the formation of the amyloid fibril by the beta-amyloid protein of Alzheimer disease. The solubility, structure and growth of the amyloid fibrils remain unaffected with the action of apoE3, implying that apoE3 is not a thermodynamic inhibitor of amyloid formation.

A study of characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter using SDS-PAGE analysis and photoaffinity cross-linking of the affinity-purified AIID2 reveals that element D has negative regulatory role on apoA-II gene transcription, when occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied by AIID2/GABP. AIID2 consists of three proteins with molecular masses 54 to 63 kDa and is homologous to GABP, an Ets-related protein. AIID1 binds to apoA-II promoter as well as regulatory element C of apoCIII, and influences their transcriptional regulation.

The use of a cloned rat editing protein cDNA hybridization probe helps clone the cDNA and genomic sequences for a human apolipoprotein B (apoB) mRNA editing protein. HPLC column and polymerization studies indicate that the apoB mRNA editing protein exists as a homodimer. Northern blot analysis reveals the exclusive expression of human apoB protein in small intestine. The cDNA, on expression in HepG2 cells, leads to editing of the intracellular apoB mRNA.

Analysis of apolipoprotein B-53 (apoB-53) and microsomal triglyceride transfer protein (MTP) expression in HeLa cells indicates that apoB-53 production depends on MTP expression. HeLa cells treated with lipids produce low flotation density and high mass apoB-53, indicating that lipids control apoB-53 production. Ultracentrifugal density flotation studies of apoB-53 reveals that apoB-53 is a high density lipoprotein.

Insulin modulates the proportion of edited apolipoprotein B (apoB) mRNA and invokes the biosynthesis and secretion of apoB-48 in rat hepatocytes. Insulin exhibits a dose-dependent effect on apoB secretion and apoB mRNA editing. Long-term exposure of primary hepatocytes to insulin helps study the influence of hyperinsulinemia on the secretion of triglyceride-rich lipoprotein by the liver.

Human apolipoprotein(a) (apo(a)) interactions with either apolipoprotein B-100 (apo B-100) or lipids do not affect apo(a) structure which is independent of the lipoprotein (a) (Lp(a)). Limited proteolysis with cathepsin D and thermolysin were used to study apo(a) interactions with apo B-100 in Lp(a) particle and its structural domains.

An analysis of apolipoprotein J (apoJ) that contains high density lipoproteins reveals its specific association with human plasma's paraoxonase, both in vitro and in vivo. The apoJ-paraoxonase mole ratio is constant at 8:2:1, validating the specificity of the apoJ-paraoxonase reaction. Introduction of apoJ into apoJ-deficient plasma causes removal of apoJ and paraoxonase by an anti-apoJ affinity region that maintains the mole ratio.

Apo(a), a low density lipoprotein similar to plasminogen, influences coronary atherosclerosis. The plasminogen gene has 5 cysteine-rich patterns called kringles, which undergo Molecular Drive-like processes that give rise to high intraspecies similarity. Application of the 'Molecular Clock Test' to the kringle-4 domain of apo(a) and plasminogen gene reveals that the segregation between the genes date back to years preceding the evolution of mammals.

Human hepatocarcinoma cell line, HepG2, is used for analyzing the role of Cys4057 in the lipoprotein(a) (Lp(a)) assembly, containing one copy of apolipoprotein B-100 and apolipoprotein (a). Analyses of cDNA plasmid expression in human hepatocarcinoma line HepG2 reveal the transformation of the amino acid sequence of apolipoprotein from Cys4057 to either serine or glycine. Experimental results corroborate the presence of a disulfide bond between Cys4057 of apolipoprotein (a) and apolipoprotein B-100, located within recombinant lipoprotein (a) particles. This bridge is vital to the integrity of lipoprotein (a)-like complex assembly which is synthesized by HepG2 cells.

A research of families and individuals with a past history of coronory heart disease indicates that the gene governing the levels of plasma apo AII has a close association with the human apo A II gene. There is also a close link between a gene regulating FFA levels and the apo AII gene. Plasma apoAII levels and plasma FFA levels are determined by a gene that is related to the apoAII gene.

Routine neuropathological reports, and analyses of Congo red- and immunostained slides are used to investigate the level of amyloid deposition in autopsy-confirmed incidents of sporadic late-onset Alzheimer disease (AD) in patients with defined apolipoprotein E genotype (APOE), to determine if the APOE genotype can be related to a characteristic neuropathological phenotype. A characteristic neuropathological phenotype of significantly enhanced vascular and plaque amyloid beta-peptide deposits compared with patients homozygous for APOE3 is evident in those with sporadic late-onset AD.

The binding of putative high density lipoprotein (HDL) receptor to apolipoprotein AII (apoAII) is examined. The reaction between HDL and its putative receptor on cells takes place. Lipid is necessary to facilitate the optimal binding of apoAII because of the association of its geometry and working. The binding reaction reveals the participation of alpha-helical areas of the lipid. The binding of apoAII to the putative receptor is independent of the state of oxidation of the net remains.

Human apolipoprotein A-II (apo A-II) has controlling regions C, D, E, F, G, I, J, and M, which bind with certain activities whose properties are determined by studying their relevance to hepatic and intestinal transcription. The synergistic interactions of elements which bind to the proximal and the distal regulatory factors govern the liver-specific revelation of the apo A-II gene. This influences the organization and characteristics of the apo A-II promoter. A single gene is controlled by the involvement of several governing nuclear factors.

The binding of synthetic amyloid beta (beta/A4) peptide was compared to purified apolipoprotein E4 (apoE4) and the most common isoform, apoE3, to find if apoE4 is related to enhanced vulnerability to Alzheimer disease. The two isoforms form a complex with the beta/A4 peptide in different ways. ApoE is regionalized in the senile plaques, congophilic angiopathy and neurofibrillary tangles of Alzheimer disease.

A study to find if lipid synthesis was really enhanced in isolated analbuminemia (NA) and to establish the regions of enhanced lipogenesis involved the estimation of the rate of cholesterol and fatty acid synthesis in liver and extrahepatic tissue in female NA and control Sprague-Dawley (SD) rats, at extrahepatic regions in NA. Significant enhancement in cholesterol and fatty acid synthesis take place. The liver is not the region of enhanced lipogensis but is a significant region of enhanced apolipoprotein synthesis,as suggested by the enhancements of apolipoprotein mRNAs encoding the principal apolipoprotein in the liver of NA rat.

Researchers studied the causes of the synthesis and secretion of apolipo proteins, including mRNA for apolipoproteins, in the human colon cancer cell line Caco-2 during differentiation in continuous culture. A competition experiment indicated that the differentiation-related intestinal factor for transcription 1 protein (DRIFT-1 protein) recognized the control areas of both the apolipoprotein A-I and B genes.

The possibility that relatively common genetic variation sites in populations of the apoCIII gene region was tested. DNA isolates and lipid profiles were derived from samples of blood and subjected to DNA sequence analysis, allele-specific oligonucleotide hybridization and maximum likelihood statistical tests. The relation of promoter polymorphism to the variant haplotypes was established.

Recombinant apolipoprotein(a) was studied by hydrodynamic and electron microscopy techniques. Recombinant apo(a) forms a tight, noncovalent complex with human low density lipoprotein (LDL). Hydrodynamic modeling suggests that it made contact with the LDL through a few kringles, with the remainder of the molecule extending into solution. This complex dissociates in the presence of 50 mM 6-aminohexanoic acid.