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M G Mulkerrin

Publications and source records attributed to M G Mulkerrin.

At least 19 recordsLinked to original sources
PubMed2001

Engineered antibodies with increased activity to recruit complement.

This manuscript describes two sites in a human IgG1 that, when mutated individually or in combination, result in a dramatic increase in C1q binding and complement-dependent cytotoxicity activity. These two residues, K326 and E333, are located at the extreme ends of the C1q binding epicenter in the C(H)2 domain of a human IgG. A mutation to tryptophan at K326 debilitates Ab-dependent cell-mediated cytotoxicity activity. In addition, substitutions of the residues E333 with serine and of K326 with tryptophan in a human IgG2 confer biological activity in the complement-dependent cytotoxicity assay in which the wild-type IgG2 is inactive. This study reveals that the residues K326 and E333 play a significant role in the control of the biological activity of an IgG molecule and can rescue the activity of an inactive IgG isotype.

Amino Acid Substitution↗
PubMed2000

Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc.

Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin's B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.

Alanine↗
PubMed1998

Contribution of domain interface residues to the stability of antibody CH3 domain homodimers.

Dimers of CH3 domains from human IgG1 were used to study the effect of mutations constructed at a domain-domain interface upon domain dissociation and unfolding, "complex stability". Alanine replacement mutants were constructed on one side of the interface for each of the sixteen interdomain contact residues by using a single-chain CH3 dimer in which the carboxyl terminus of one domain was joined to the amino terminus of the second domain via a (G4S)4 linker. Single-chain variants were expressed in Escherichia coli grown in a fermentor and recovered in yields of 6-90 mg L-1 by immobilized metal affinity chromatography. Guanidine hydrochloride-induced denaturation was used to follow domain dissociation and unfolding. Surprisingly, the linker did not perturb the complex stability for either the wild type or two destabilizing mutants. The CH3 domain dissociation and unfolding energetics are dominated by six contact residues where corresponding alanine mutations each destabilize the complex by >2.0 kcal mol-1. Five of these residues (T366, L368, F405, Y407, and K409) form a patch at the center of the interface and are located on the two internal antiparallel beta-strands. These energetically key residues are surrounded by 10 residues on the two external beta-strands whose contribution to complex stability is small (three have a Delta DeltaG of 1.1-1.3 kcal mol-1) or very small (seven have a Delta DeltaG of </=0.7 kcal mol-1). Thus, at the center of the CH3 structural interface there is a small "functional interface" of residues that make significant contributions to complex stability.

Alanine↗
PubMed1997

Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion.

An HPLC procedure was validated for determining the purity with respect to the charge variant distribution of the recombinant monoclonal antibody (MAb) IDEC-C2B8 by high-performance ion-exchange chromatography. Papain was used to fragment the molecule into Fab and Fc fragments prior to chromatographic analysis. Fragmentation allowed the resolution of the variants arising from the cyclization of glutamine to pyroglutamate at the amino-terminus of the light and heavy chains (Fab-pE/Q variants) from the variants resulting from the processing of the carboxy-terminal lysine residues of the heavy chains (Fc-Lys variants). The assay demonstrated good linearity, yielding correlation coefficients of > 0.99 for total protein, Fc-Lys variants and Fab-pE/Q variants. Recovery of total protein from the column was 95.7%. Sample recovery studies demonstrated a mean accuracy of 102% for a Fab fragment over the range 2-10% of the total protein. The limit of detection was 0.2 microg and 0.1 microg for Fc and Fab variants, respectively. The repeatability of the assay and intermediate precision had relative standard deviation (RSD) values of < 1%. Parameters of the papain digest (time, digest stability, reagent stability, pH and papain vendor) and of the chromatography (mobile phase pH, stability, buffer concentration, and column lot and aging) were evaluated for robustness and determined to be acceptable. Data are presented demonstrating the suitability of the assay for determining the product purity of a recombinant MAb.

Antibodies, Monoclonal↗
PubMed1997

Rapid analytical tryptic mapping of a recombinant chimeric monoclonal antibody and method validation challenges.

A rapid and reproducible analytical tryptic mapping method was developed as an identity test for a recombinant chimeric monoclonal antibody for lot release testing. The unfolding, reduction, carboxymethylation, trypsin digestion, and reversed-phase (RP) HPLC steps were optimized to provide a reproducible method. The optimized method requires 30 min for unfolding the protein, 30 min for carboxymethylation, 4 h for digestion with TPCK-trypsin and 140 min for RPHPLC analysis. The total time required is less than 8 h compared to conventional procedures, which must be performed over several days. The optimized method was validated for its precision, recovery, specificity, and robustness. The precision of the method was determined by repeatability and intermediate precision experiments. Relative standard deviation (RSD) values were < or = 10% for the relative peak areas of marker peaks. The mean recovery of these marker peaks was 88.4%. The specificity was demonstrated by the unique tryptic mapping patterns obtained compared with several other monoclonal antibodies. Robustness was demonstrated by the relative insensitivity of the tryptic map to small deliberate changes in key method parameters. Excessive relative peak area variability observed for one peak (RSD 52%) was traced to adsorption to glass autosampler vials. This variability was substantially reduced (RSD 11%) by substituting polypropylene autosampler vials. The data demonstrate that this method may be applicable to a wide range of pharmaceutically relevant monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1995

Ligand binding to the tissue-type plasminogen activator kringle 2 domain: structural characterization by 1H-NMR.

Ligand binding to a recombinant human tissue-type plasminogen (tPA) kringle 2 domain has been characterized via 1H-NMR spectroscopy at 500 MHz. Seven omega-amino acid ligands were investigated: L-Lys, 6-aminohexanoic acid (6AHA), 7-aminoheptanoic acid (7AHA), trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA), p-(aminomethyl)benzoic acid (PAMBA), p-(aminoethyl)benzoic acid (PAEBA), and p-benzylaminesulfonic acid (BASA). The interactions with two peptides containing a C-terminal lysyl residue, Tyr-Leu-Leu-Lys (YLLK) and Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), were also studied. The sequence AFQYHSK is found within the plasminogen N-terminal activation peptide while the tetrapeptide YLLK corresponds the 119-122 segment of the fibrinogen B beta-chain. Spectral comparison of ligand-free and ligand-containing kringle 2 samples leads to the conclusion that all the small ligands as well as the peptides' C-terminal lysyl residues interact with a common binding site in kringle 2. Two-dimensional spectra show that besides the Tyr36, Trp62, His64, Trp72, and Tyr74 aromatic rings, the Val35 and Asp55 aliphatic side chains also participate in ligand binding. Contact points with the ligands 6AHA and BASA were unambiguously identified from kringle 2-ligand nuclear Overhauser effects (NOEs). Overall, the ligand-induced chemical shifts and the intermolecular NOEs correlate remarkably well. Association constant (Ka) values for the kringle 2-ligand interactions were determined. Among the investigated ligands, BASA perturbs the kringle 2 spectrum the most and exhibits the highest affinity for kringle 2 (Ka approximately 233 mM-1). Of the two other aromatic ligands, PAEBA binds to kringle 2 less firmly (Ka = approximately 12 mM-1) than does the one-methylene group shorter analog PAMBA (Ka approximately 31 mM-1). By comparison, relative to 6AHA (Ka approximately 22 mM-1), the longer chain linear aliphatic ligand 7AHA interacts with kringle 2 with significantly higher affinity (Ka approximately 149 mM-1). By reference to the NMR-derived binding site structure, it is suggested that the higher affinity toward 7AHA may stem from (a) a relatively more favored ionic pairing between its carboxylate group and the LYs34 + Arg 69 side-chain cationic centers and (b) an enhanced interaction between the ligand hydrocarbon moiety and the kringle hydrophobic pocket, in particular with the Leu70 side chain. The latter is consistent with the relatively good affinity of kringle 2 for the cyclic hydrocarbon ligand AMCHA (Ka approximately 69 mM-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Conformational changes in the reversed phase liquid chromatography of recombinant human growth hormone as a function of organic solvent: the molten globule state.

As a continuation of a previous paper on the retention behavior of recombinant human growth hormone (rhGH) in reversed phase chromatography at pH 6.5 (Oroszlan, P., et al. Anal. Chem. 1992, 64, 1623-1631) the effect of 1-propanol (1-PrOH) and acetonitrile on the conformation of rhGH at this pH has been investigated by circular dichroism (CD), second-derivative UV spectroscopy, fluorescence anisotropy, fluorescence quenching, and fluorescence lifetime measurements. Addition of 1-PrOH up to a concentration of 10% (v/v) does not cause any significant changes in protein structure. However, above this concentration, a transition from the native to a new state is observed; the transition is completed above 30% (v/v) of 1-PrOH, the composition for completion being dependent on temperature. This change in structure correlates with retention changes observed in reversed phase chromatography. The new rhGH conformation retains much of the alpha-helicity and possesses a slightly expanded hydrodynamic radius relative to native rhGH. Second-derivative UV spectroscopy suggests that the hydrogen bond between Trp 86 and Asp 169, spanning two alpha-helices, remains intact. On the other hand, the near-UV CD intensity changes from positive to negative in the Trp region of the spectrum, signaling an alteration in the Trp environment. In addition, fluorescence quenching measurements with trichloroethanol reveal greater accessibility to solvent of the Trp residue after the conformational transition has occurred. From the results, it is concluded that a molten globule state (compact state retaining much of the secondary structure of the native state but with a disrupted tertiary structure) is produced with the addition of > 30% (v/v) 1-PrOH.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Propanol↗
PubMed1994

Contribution to global protein stabilization of the N-capping box in human growth hormone.

In this work we have investigated the contribution to protein stability of residues forming the boundaries of alpha-helices. At the N-terminus of helix 2 of human growth hormone there are two residues, Ser71 and Glu74, which form two reciprocal hydrogen bonds between the side chains and the backbone nitrogens of either residue (the N-capping box). In order to evaluate the stabilizing effect of each hydrogen bond, site-directed mutagenesis was employed. In addition, the effect of side-chain negative charge on helix stabilization, via charge dipole interaction, was assessed. Ultraviolet spectroscopy and near- and far-UV CD spectroscopy as well as guanidine hydrochloride protein denaturation were used as assays to monitor the conformational and free energy of stabilization changes induced by the point mutations. The results of these experiments can be summarized as follows: (a) receptor binding studies showed that the tertiary conformation of each mutant was similar to that of the native hormone, (b) far-UV CD spectroscopic analyses showed that the overall alpha-helical content was unchanged in the mutants, (c) UV absorption and CD spectroscopic analyses indicated small alterations in helical packing in those mutants in which the hydrogen bond between the side chain of Ser71 and backbone NH of Glu74 was disrupted, (d) the hydrogen bond involving the side chain of Ser71 contributes at least 1.0 kcal/mol to protein stabilization and has a 2-fold larger stabilizing effect than that of the hydrogen bond involving the Glu74 side chain, and (e) the putative charge-dipole interaction of Glu74 with the alpha-helix dipole does not contribute to the stabilization of the tertiary conformation of human growth hormone.

Circular Dichroism↗
PubMed1993

The molecular basis for growth hormone-receptor interactions.

High-resolution mutational and structural analyses of purified components have revealed a great deal about the molecular basis for growth hormone action. The structural and functional aspects of the interactions between hGH and its receptors have been largely elaborated. From these studies it has been possible to engineer homologues of hGH to bind to the hGH receptor and act as potential antagonists. Receptor-selective and high-affinity analogs have also been constructed based on a combination of alanine scanning and monovalent phage display. From this molecular work much has been revealed about the biology of hGH (Fig.9). Our data suggest that hGH is stored in the pituitary as a (Zn2+,hGH)2 complex. On release from somatotropic vesicles it dissociates into a monomeric form and reveals its primary receptor binding site (site 1). Free hGH can bind to the hGHbp in serum to form monomeric or dimeric complexes that slow the clearance of hGH (Moore et al., 1989). However, because the affinity for the full-length receptor is greater, hGH can bind to it preferentially. Furthermore, the constitutive levels of the hGHbp (approximately 0.5 to 1 nM) (Baumann et al., 1986; Herrington et al., 1986) are considerably below the levels of hGH after pulsatile release (approximately 2 to 5 nM) (Thompson et al., 1972). Our data indicate that hGH binds to the hGH receptor on cell membranes through site 1 and subsequently forms dimers through site 2. We believe a similar process may occur for hGH to activate the hPRL receptor, except that Zn2+ is required for site 1 association. Such receptor dimers are then activated and capable of interacting with other cellular components that may mediate the hGH "signal." Recently, based upon this proposed mechanism, we produced potent antagonists to the hGH receptor (Fuh et al., 1992) and hPRL receptor (G. Fuh, P. Colosi, W. Wood, and J. Wells, unpublished results). These antagonists bind tightly to site 1 but are blocked in their ability to bind site 2 and dimerize the receptor. We believe these methods and discoveries will be relevant to the study of signaling by other hematopoietic hormones and receptors as well as other hormones and receptors.

Amino Acid Sequence↗
PubMed1992

Mambin, a potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor structurally related to the short neurotoxins.

The purification, complete amino acid sequence, functional activity, and structural modeling are described for mambin, a platelet glycoprotein GP IIb-IIIa antagonist and potent inhibitor of platelet aggregation from the venom of the Elapidae snake Dendroaspis jamesonii (Jameson's mamba). Mambin is 59 residues in length and contains four disulfide linkages and an RGD amino acid sequence found in protein ligands that bind to GP IIb-IIIa. Mambin inhibits ADP-induced platelet aggregation (IC50 = 172 +/- 22 nM) and inhibits the binding of purified platelet fibrinogen receptor GP IIb-IIIa to immobilized fibrinogen (IC50 = 3.1 +/- 0.8 nM). Mambin has very little sequence similarity to the Viperidae family of platelet aggregation inhibitors, except for the RGD-containing region in the protein. However, mambin does have ca. 47% similarity to the short-chain postsynaptic neurotoxins found in other Elapidae venoms, which do not contain the RGD sequence and do not act as GP IIb-IIIa antagonists. On the basis of its circular dichroism spectrum, mambin has a beta-sheet structure characteristic of the neurotoxins. Molecular modeling of the mambin sequence onto the erabutoxin b structure predicts a very similar structure within the entire protein except for the loop containing the RGD sequence. Mambin may therefore represent a genetic hybrid of neurotoxic and hemotoxic proteins found in snake venoms.

Amino Acid Sequence↗
PubMed1991

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule.

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.

Amino Acid Sequence↗
PubMed1991

Dimerization of human growth hormone by zinc.

Size-exclusion chromatography and sedimentation equilbrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn(2+)-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn(2+)-hGH dimeric complex may be important for storage of hGH in secretory granules.

Amino Acid Sequence↗
PubMed1991

A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor.

A mutational strategy is presented that allowed us to identify hormone-binding determinants in the extracellular portion of the human growth hormone receptor (hGHbp), a 238-residue protein with sequence homology to a number of cytokine receptors. By systematically replacing side chains with alanine we probed the importance of charged residues (49 total, typically located on the surface), aromatic residues (9 total), and neighbors of these (26 total). The alanine substitutions that were most disruptive to hormone binding are located predominantly in four segments of a cysteine-rich domain in the hGHbp, and collectively they form a patch when mapped upon a structural model proposed for cytokine receptors. Control experiments with monoclonal antibodies confirmed that most of these alanine substitutions do not disrupt the overall antigenic structure of the hGHbp. This high-resolution functional analysis will complement structural studies and provides a powerful basis for evaluating and engineering the energetics of hormone-receptor interactions. Moreover, the hormone-binding determinants identified here may be similarly located in other, homologous, receptors.

Alanine↗
PubMed1990

Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments.

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig VL domains. The first of these (V1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig VL domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V1 and V2 CD4 domains; this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig VL domains; this directly supports the hypothesis that the V1 and V2 domains of CD4 fold similarly to Ig VL domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain.

A gene fragment encoding the extracellular domain of the human growth hormone (hGH) receptor from liver was cloned into a plasmid under control of the Escherichia coli alkaline phosphatase promoter and the heat-stable enterotoxin (StII) signal peptide sequence. Strains of E. coli expressing properly folded hGH binding protein were identified by blotting colonies with 125I-hGH. The E. coli strain capable of highest expression (KS330) secreted 10 to 20 mg/liter of culture of properly processed and folded hGH receptor fragment into the periplasmic space. The protein was purified to near homogeneity in 70 to 80% yield (in tens of milligram amounts) using ammonium sulfate precipitation, hGH affinity chromatography, and gel filtration. The unglycosylated extracellular domain of the hGH receptor has virtually identical binding properties compared to its natural glycosylated counterpart isolated from human serum, suggesting glycosylation is not important for binding of hGH. The extracellular binding domain codes for 7 cysteines, and we show that six of them form three disulfide bonds. Peptide mapping studies show these disulfides are paired sequentially to produce short loops (10-15 residues long) as follows: Cys38-Cys48, Cys83-Cys94, and Cys108-Cys122. Cys241 is unpaired, and mutagenic analysis shows that the extreme carboxyl end of the receptor fragment (including Cys241) is not essential for folding or binding of the protein to hGH. High level expression of this receptor binding domain and its homologs in E. coli will greatly facilitate their detailed biophysical and structural analysis.

Amino Acid Sequence↗
PubMed1989

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons.

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli.

We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence. A large portion of the secreted protein is associated with an insoluble cellular fraction. This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione. Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography. The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed. Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen [Castellino et al. (1986) Arch. Biochem. Biophys. 247, 312-320]. Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM. Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids [Winn et al. (1980) Eur. J. Biochem. 104, 579-586], we have shown the lysine binding site of t-PA kringle-2 to have a preference for a ligand with 8.8-A separation between amine and carboxylate functions. Charge interactions with the epsilon-amino group of L-lysine are important in binding since the affinities for N epsilon-acetyl-L-lysine, L-arginine, and gamma-guanidinobutyric acid are decreased greater than 2000-fold, 200-fold, and 12-fold, respectively, relative to the affinity for L-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗