Effect of zinc, copper, and calcium on the structure and stability of serum amyloid A

Serum amyloid A (SAA) is a highly conserved acute phase reactant protein, and its concentration in serum can increase up to approximately 1000 times after an inflammatory stimuli. SAA is mainly associated with high-density lipoproteins in serum, and its main function appears to involve cholesterol transport and lipid metabolism. However, SAA has also been associated with many other functions and a number of diseases, although these potential links remain poorly understood. The three-dimensional structure of SAA is not known, but we have shown that murine SAA2.2 can exist in solution as a marginally stable hexamer, which at 37 degrees C dissociates to a monomeric species that misfolds irreversibly and self-assembles into amyloid fibrils. Thus, the structure and function of SAA in vivo appear to be modulated when it binds to other proteins or small ligands. Herein, the effect of copper (Cu2+), zinc (Zn2+), and calcium (Ca2+) on the structure and stability of SAA2.2 in aqueous solution was examined using various probes of quaternary, tertiary, and secondary structure. At different concentrations of metals, including those found in the serum, the results show that the structure and stability of SAA2.2 are differently affected depending on the metal type and concentration. Copper (10-100 microM) was found to shift the equilibrium from hexamer to monomer without affecting significantly the stability of the tertiary and secondary structure of SAA2.2. In contrast, zinc (1-10 microM) bound to SAA2.2 and stabilized its quaternary, tertiary, and secondary structure. Calcium (1-10 mM) destabilized all elements of SAA2.2 structure and induced its aggregation at 10 mM. Complete aggregation of SAA2.2 was also observed when it was incubated with 1 mM Cu2+ or Zn2+, further demonstrating the tenuous structure and stability of SAA2.2. Thus, these results suggest that the many functional and pathological roles attributed to SAA may rely on its precarious structure, modulated by its interaction with ligands under homeostasis conditions and during the acute phase response.     

Serum amyloid A 2.2 refolds into a octameric oligomer that slowly converts to a more stable hexamer

Serum amyloid A (SAA) is an inflammatory protein predominantly bound to high-density lipoprotein in plasma and presumed to play various biological and pathological roles. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a marginally stable hexamer at 4–20 °C, but becomes an intrinsically disordered protein at 37 °C. Here we show that when urea-denatured SAA2.2 is dialyzed into buffer (pH 8.0, 4 °C), it refolds mostly into an octameric species. The octamer transitions to the hexameric structure upon incubation from days to weeks at 4 °C, depending on the SAA2.2 concentration. Thermal denaturation of the octamer and hexamer monitored by circular dichroism showed that the octamer is ∼10 °C less stable, with a denaturation mid point of ∼22 °C. Thus, SAA2.2 becomes kinetically trapped by refolding into a less stable, but more kinetically accessible octameric species. The ability of SAA2.2 to form different oligomeric species in vitro along with its marginal stability, suggest that the structure of SAA might be modulated in vivo to form different biologically relevant species.     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

Urea-induced denaturation of apolipoprotein serum amyloid A reveals marginal stability of hexamer

Serum Amyloid A (SAA) is an acute phase reactant protein that is predominantly found bound to high-density lipoprotein in plasma. Upon inflammation, the plasma concentration of SAA can increase dramatically, occasionally leading to the development of amyloid A (AA) amyloidosis, which involves the deposition of SAA amyloid fibrils in major organs. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a hexamer containing a central channel. Here we show using various biophysical and biochemical techniques that the SAA2.2 hexamer can be totally dissociated into monomer by approximately 2 M urea, with the concerted loss of its alpha-helical structure. However, limited trypsin proteolysis experiments in urea showed a conserved digestion profile, suggesting the preservation of major backbone topological features in the urea-denatured state of SAA2.2. The marginal stability of hexameric SAA2.2 and the presence of residual structure in the denatured monomeric protein suggest that both forms may interconvert in vivo to exert different functions to meet the various needs during normal physiological conditions and in response to inflammatory stimuli.     

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.     

Influence of the carboxy terminus of serum amyloid A on protein oligomerization, misfolding, and fibril formation

The fibrillar deposition of serum amyloid A (SAA) has been linked to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform, SAA2.2, from the CE/J mouse strain, as a model system to explore the inherent structural and biophysical properties of SAA. Despite its nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in vitro, suggesting that SAA proteins are inherently amyloidogenic. However, whereas the importance of the amino terminus of SAA for fibril formation has been well documented, the influence of the proline-rich and presumably disordered carboxy terminus remains poorly understood. To clarify the inherent role of the carboxy terminus in the oligomerization and fibrillation of SAA, we truncated the proline-rich final 13 residues of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal truncated SAA2.2 (SAA2.2ΔC) did not oligomerize to a hexamer or octamer, but formed a high molecular weight soluble aggregate. Moreover, SAA2.2ΔC also exhibited a pronounced decrease in the rate of fibril formation. Intriguingly, when equimolar amounts of denatured SAA2.2 and SAA2.2ΔC were mixed and allowed to refold together, the mixture formed an octamer and exhibited rapid fibrillation kinetics, similar to those for full-length SAA2.2. These results suggest that the carboxy terminus of SAA, which is highly conserved among SAA sequences in all vertebrates, might play important structural roles, including modulating the folding, oligomerization, misfolding, and fibrillation of SAA.     

Thermal transitions in serum amyloid A in solution and on the lipid: implications for structure and stability of acute-phase HDL

Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma HDL. SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace a substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the nonexchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies in acute-phase HDL. Second, we report an α-helix folding/unfolding transition in SAA in the presence of lipid at near-physiological temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.     

A hepatocyte receptor for high-density lipoproteins specific for apolipoprotein A-I

Apolipoprotein (apo) E-deficient rat high-density lipoproteins (HDL) bind to isolated rat hepatocytes at 4 degrees C by a process shown to be saturable and competed for by an excess of unlabeled HDL. Uptake (binding and internalization) at 37 degrees C was also saturable and competed for by an excess of unlabeled HDL. At 37 degrees C the HDL apoprotein was degraded as evidenced by the appearance of trichloroacetic acid-soluble radioactivity in the incubation media. The binding of a constant amount of 125I-apo-E-deficient HDL was measured in the presence of increasing concentrations of various lipoproteins. HDL and dimyristoyl phosphatidylcholine (DMPC) X apo-A-I complexes decreased binding by 80 and 65%, respectively. Human low-density lipoproteins, DMPC X apo-E complexes, and DMPC vesicles alone did not compete for apo-E-deficient HDL binding. However, DMPC X apo-E complexes did compete for the binding of the total HDL fraction that contained apo-E but to a lesser extent than did DMPC X apo-A-I. DMPC X 125I-apo-A-I complexes also bound to hepatocytes, and this binding was competed for by excess HDL (70%) and DMPC X apo-A-I complexes (65%), but there was no competition for binding by DMPC vesicles or DMPC X apo-E complexes. It thus appears that hepatocytes have a specific receptor for HDL and that apo-A-I is the ligand for this receptor.     

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.     

The effect of temperature and Ca2+ on the interaction of high-density lipoproteins with epithelial cells in the human small intestine

The effect of temperature and Ca2+ ions on the interaction of high-density lipoprotein (HDL3) with human enterocytes was studied. It was shown that Kd measured at 4 degrees C is similar to that at 37 degrees C. Maximal number of binding sites at 37 degrees C is 15-fold times higher than that at 4 degrees C. EDTA (10 mM) and CaCl2 (0.5-5 mM) did not affect binding and uptake of HDL3 by human enterocytes. The obtained results indicate that HDL3-binding with these cells depends on temperature and does not depend on Ca2+ ions.     

Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA

The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.     

High-density lipoprotein binding to guinea-pig hepatic membranes. Comparison of guinea-pig and human ligands

Human and guinea-pig apo-E-free HDL were incubated with isolated guinea-pig hepatic membranes at 4 and 37 degrees C to determine the effects of temperature and heterologous systems on the equilibrium parameters of HDL binding. Receptor mediated HDL binding was highest at 37 degrees C for both lipoproteins. At 4 degrees C, a higher affinity constant (Kd) was observed when guinea-pig HDL was the ligand relative to human HDL; in contrast, both HDL preparations had similar Kd values at 37 degrees C. Calculated binding and receptor number (Bmax) were higher at both temperatures when guinea-pig HDL was the ligand. These results demonstrate a significant species difference in HDL binding to hepatic membrane which is partially temperature-dependent.     

Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake

Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that (125)I-labeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30-50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.     

The N-terminus and C-terminus of IFN-gamma are binding domains for cloned soluble IFN-gamma receptor.

The mechanism of binding of murine IFN-gamma to its receptor has not been determined. We have studied this mechanism by examining the binding of overlapping synthetic peptides of IFN-gamma to cloned soluble murine IFN-gamma R. IFN-gamma (1-39) and IFN-gamma (95-133) were able to compete with [125I]IFN-gamma for binding to cloned soluble receptor. Peptides corresponding to the inner region of IFN-gamma--IFN-gamma (36-60), IFN-gamma (54-91), and IFN-gamma (78-107)--showed a markedly reduced ability to compete with [125I]IFN-gamma for receptor binding relative to the N-terminal and C-terminal peptides. In direct binding studies, the binding of [125I]-IFN-gamma (1-39) to soluble receptor could only be competed by IFN-gamma (1-39) and IFN-gamma and not by any of the other peptides including IFN-gamma (95-133). This suggests that the N- and C-termini of IFN-gamma bind to different regions of the receptor. These data in conjunction with previous structure/function studies and x-ray crystallographic data have allowed us to formulate a "velcro-key" model of IFN-gamma binding to receptor that involves both the N- and C-terminal domains. The N-terminus binds in the classical "lock-and-key" manner characterized by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct from the N-terminus likely through the polycationic region, which is conserved across species barriers. Binding of this type would exhibit high affinity and low specificity similar to a piece of velcro. This interaction becomes specific when the C-terminus is in the context of the whole IFN-gamma molecule and may act to increase the affinity of receptor binding and/or facilitate signal transduction.     

Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages

Iodinated colony-stimulating factor produced by L-cells (125I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37 degrees C, the cell-bound 125I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0 degree C, the cell-bound 125I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37 degrees C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH4Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added 125I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37 degrees C but not at 0 degrees C in culture medium containing fetal calf serum.     

Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding

A mammalian vesicular neurotransmitter transporter has been expressed in the yeast Saccharomyces cerevisiae. The gene encoding the rat vesicular monoamine transporter (rVMAT(1)) was cloned in several expression plasmids. The transporter was expressed at detectable levels only when short sequences using codons favored by S. cerevisiae were fused preceding the start of translation of rVMAT(1). The scarce expression of the wild-type protein was, most likely, due to the fact that part of the N-terminus of the protein is encoded by codons not preferred in S. cerevisiae. Furthermore, low growth temperatures increased rVMAT(1) expression and altered its processing. Whereas at 30 degrees C the protein is not glycosylated, at lower temperatures ( approximately 16 degrees C) half of the expressed transporters undergo core glycosylation. In addition, under these conditions the levels of protein expression significantly increase. Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT(2)) was also expressed in yeast. The yeast-expressed bVMAT(2) binds [(3)H]dihydrotetrabenazine ([(3)H]TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules. Dodecyl maltoside-solubilized bVMAT(2) retains the conformation required for [(3)H]TBZOH binding. We exploited the robust binding to follow the transporter during purification assays on a Ni(2+)-chelating column. In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S. cerevisiae.     

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.     

Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein,serum amyloid A 2.1

We show that murine macrophages that have ingested cell membranes as a source of cholesterol exhibit a marked increase in acyl-CoA:cholesterol acyl transferase (ACAT) activity. Exposure of these macrophages to acute-phase high-density lipoprotein (HDL) results in a marked reduction of ACAT and enhancement of cholesteryl ester hydrolase (CEH) activities, phenomena not seen with native HDL. These complementary but opposite effects of acute-phase HDL on the two enzyme systems that regulate the balance between esterified (storage) cholesterol and unesterified (transportable) cholesterol are shown to reside with serum amyloid A (SAA) 2.1, an acute-phase apolipoprotein of HDL whose plasma concentration increases 500- to 1,000-fold within 24 h of acute tissue injury. Mild trypsin treatment of acute-phase HDL almost completely abolishes the apolipoprotein-mediated effects on the cholesteryl ester cycle in cholesterol-laden macrophages. The physiological effect of SAA2.1 on macrophage cholesterol is to shift it into a transportable state enhancing its rate of export, which we confirm in tissue culture and in vivo. The export process is shown to be coupled to the ATP binding cassette transport system. Our findings integrate previous isolated observations about SAA into the sphere of cholesterol transport, establish a function for a major acute-phase protein, and offer a novel approach to mobilizing macrophage cholesterol at sites of atherogenesis.     

Low temperature inhibition on binding, trasport, and incorporation of leucine, arginine, methionine, and histidine in Escherichia coli).

A multiple amino acid auxotroph and a wild type of Escherichia coli K12 were used to study the effects of near minimum growth temperatures on the binding, transport, and cellular incorporation of selected amino acids. Both strains of the bacterium showed the same minimum growth temperature (8 degrees C) when previously grown at 15 degrees C. At 8 degrees C and above, the auxotroph exhibited an overall greater ability to bind and transport amino acids than did the wild type. Below the minimum growth temperature, transport and cellular incorporation including respiration ((uptake) were significantly lower for either organism. The NEU and HEPPEL osmotic shock treatment indicated the removal of the specific histidine-binding protein and the ability to bind histidine was not recovered by further incubation below 8 degrees C. At 8 degrees C and above, the cells recovered their ability to bind histidine within one hour. The evidence presented indicates a direct relationship between the auxotroph's minimum growth temperature and its ability to bind amino acids, specifically methionine.