Precursor activation in a pyoverdine biosynthesis.

The siderophore produced by Azotobacter vinelandii strain UW belongs to a large family of peptidic siderophores collectively called pyoverdines. The biosynthesis of the peptidyl moiety of this siderophore was shown to involve activation of the constituent amino acids as their adenylates, as demonstrated by amino acid-dependent ATP-[32P]pyrophosphate exchange. The enzyme system responsible for this activation was partially purified by chromatographic techniques.

Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1.

Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both pyruvate oxidation and lysine: N6-hydroxylation were inhibited by protonophores and Gramicidin S.

Spin label EPR structural studies of the N-terminus of alpha-spectrin.

Spectrin, a vital component in human erythrocyte, is composed of alpha- and beta-subunits, which associate to form (alphabeta)2 tetramers. The tetramerization site is believed to involve the alpha-spectrin N-terminus and the beta-spectrin C-terminus. Abnormal interactions in this region may lead to blood disorders. It has been proposed that both termini consist of partial structural domains and that tetramerization involves the association of these partial domains. We have studied the N-terminal region of a model peptide for alpha-spectrin by making a series of double spin-labeled peptides and studying their dipolar interaction by electron paramagnetic resonance methods. Our results indicate that residues 21-42 of the N-terminus region exhibit an alpha-helical conformation, even in the absence of B-spectrin.

Characterization of the pyoverdines of Azotobacter vinelandii ATCC 12837 with regard to heterogeneity.

Azotobacter vinelandii strain ATCC 12837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows that A. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to the Azotobacter-type family of pyoverdines.

The importance of the hydrophobic components of the binding energies in the interaction of omega-amino acid ligands with isolated kringle polypeptide domains of human plasminogen.

Three of the five kringle domains of human plasminogen (HPg), viz the first, fourth and fifth, exhibit significantly strong binding to omega-amino acids, such as epsilon-aminocaproic acid (EACA) and transaminomethylcyclohexane-1-carboxylic acid (AMCHA). In all cases, ligand stabilization is due to ion dipole attractions of its charged groups with polypeptide side chains, as well as hydrophobic clustering of the ligand methylene groups with appropriate hydrophobic residues within the kringle domain. In order to estimate the significance of the hydrophobic components of ligand stabilization, we have sought a more detailed description of these binding interactions. The standard thermodynamic binding parameters, delta G degrees, delta H degrees and delta S degrees, for association of EACA and AMCHA with isolated recombinant kringle regions of HPg have been determined at several temperatures to evaluate the changes in standard heat capacities (delta C degrees p) accompanying these interactions. In each case, the delta C degrees p values of binding were negative and in the range -36 to -91 cal mol -1 K -1, reflective of the importance of the hydrophobic components of the binding process and their probable effects on surrounding water structure.

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.

Flexibility of the alpha-spectrin N-terminus by EPR and fluorescence polarization.

The structure and flexibility of the biologically important alpha-spectrin amino terminal region was examined by the use of fluorescence and EPR spectroscopy. The region studied has been previously demonstrated to be essential for the alpha-spectrin:beta-spectrin association of the tetramerization site. Appropriate spectroscopic probe moieties were coupled to this region in a recombinant fragment of human erythroid alpha-spectrin. There was good agreement between the EPR and fluorescence techniques in most of this region. Mobility determinations indicated that a portion of the region was relatively immobilized. This is significant, since although predictive methods have indicated that this region should be alpha-helical, previous experimental evidence obtained on smaller synthetic peptides had indicated that this region was disordered. Observed rigidity appears to be incompatible with such a disordered state, and has important ramifications for the flexibility of this molecule that is so integral to its role in stabilizing erythrocyte membranes.

Amino acid residues of the kringle-4 and kringle-5 domains of human plasminogen that stabilize their interactions with omega-amino acid ligands.

Regions of the human plasminogen (Pg) cDNA containing its kringle 4 (K4) and K5 domains have been expressed in Escherichia coli, and binding constants of omega-amino acid ligands for recombinant (r)-[K4Pg] and r-[K5Pg] have been obtained. In each case, the results showed that of a series of aliphatic alpha, omega-amino acid analogues, 6-aminohexanoic acid showed maximal affinity for these modules, and all ligands interacted more strongly with r-[K4Pg] than with r-[K5Pg]. Site-directed mutagenesis investigations demonstrated that the major amino acid side chain contributors to ligand binding were similar for each of these kringles. Ligand binding was stabilized by charged groups at Asp56 of r-[K4Pg] and Asp57 of r-[K5Pg] as well as by Arg69 of both r-[K4Pg] and r-[K5Pg]. Some hydrophobic amino acids that contributed significantly to the binding strength of the ligands were identified. These were provided by homologous residues in each of the domains, viz. Trp60 and Trp70 of r-[K4Pg] and Trp62 and Tyr72 of r-[K5Pg]. Tyr74 of r-[K5Pg] also substantially contributed to its ligand binding energy.

Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization.

We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization. A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of alpha-spectrin. Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in alpha-spectrin. The alpha-spectrin DNA fragment and a DNA fragment containing the last 449 residues in beta-spectrin were introduced into the yeast two-hybrid system for rapid screening of alpha- and beta-spectrin interaction. Yeast colonies with interacting alpha- and beta-peptides were blue, and those with non-interacting alpha- and beta-peptides were white. Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of alpha- and beta-spectrin at the tetramerization site.

Interactions of the alpha-spectrin N-terminal region with beta-spectrin. Implications for the spectrin tetramerization reaction.

Spectrin of the erythrocyte membrane skeleton is composed of alpha- and beta-spectrin, which associate to form heterodimers and tetramers. It has been suggested that a fractional domain (helix C) in the amino-terminal region of alpha-spectrin (Nalpha region) bundles with another fractional domain in the carboxyl-terminal region of beta-spectrin (Cbeta region) to yield a triple alpha-helical bundle and that this helical bundling is largely responsible for tetramer formation. However, there are certain objections to assigning a preeminent role to this helical bundling in the tetramerization reactions. We prepared several recombinant peptides of alpha-spectrin fragments spanning only the Nalpha region (lacking the dimer nucleation site) and quantitatively studied their interaction with beta-spectrin. We found that a majority of the interactions were localized, as expected, in the Nalpha-helix C region but that there was also some contribution from the nonhomologous region. More importantly, the temperature and ionic strength dependence of this interaction in our model peptides was different from that in intact spectrin. We suggest that, although the regions involving the putative helical bundling in alpha- and beta-spectrin undoubtedly play a significant role in tetramerization, regions distal to the Nalpha-helix C region in spectrin are also involved in tetramer formation. Structural flexibility and lateral interactions may play a role in spectrin tetramerization.

Amino acids of the recombinant kringle 1 domain of human plasminogen that stabilize its interaction with omega-amino acids.

A series of strategically designed recombinant (r) mutants of the kringle 1 region of human plasminogen ([K1HPg]) have been constructed and the resulting gene products employed to reveal the identities of the residues that contribute to stabilization of the binding of omega-amino acid ligands to this domain. On the basis of determinations of the binding constants of the ligands, 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, to a variety of these mutants, we find that the anionic site of the polypeptide responsible for stabilization of the amino group of the ligands consists of both D54 and D56 and the cationic site of the polypeptide that interacts with the carboxylate group of the ligand is composed solely of R70. The main hydrophobic interactions that stabilize binding of these ligands, likely by interactions with the ligand hydrophobic regions, are principally due to W61, Y63, and Y71. The results obtained are consistent with conclusions that could be made from analysis of the X-ray crystal structure of r-[K1HPg] and from previous studies from this laboratory regarding the binding of ligands of this type to the kringle 2 region of tissue-type plasminogen activator ([K2tPA]). It thus appears as though a common ligand binding site has evolved in different kringles with ligand specificity differences between r-[K2tPA] and r-[K1HPg] perhaps explainable by the different nature of the cationic sites on these polypeptides that are involved in coordination to the ligand carboxylate groups.

Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids.

An Escherichia coli expression vector, containing the alkaline phosphatase promoter and the stII heat-stable enterotoxin signal sequence, along with the cDNA of the kringle 1 (K1) region of human plasminogen (HPg), has been employed to express into the periplasmic space amino acid residues 82-163 (E163----D) of HPg. This region of the molecule contains the entire K1 domain (residues C84-C162) of HPg, as well as two non-kringle amino-terminal amino acids (S82-E83) that are present in their normal locations in HPg and a carboxyl-terminal amino acid, D163, that results from mutation of the E163, normally present at this location in the HPg amino acid sequence. After purification of r-K1 by chromatographic techniques, we have investigated its omega-amino acid binding properties by titration calorimetry, intrinsic fluorescence, and differential scanning microcalorimetry (DSC). The antifibrinolytic agent, epsilon-aminocaproic acid (EACA), possesses a single binding site for r-K1. The thermodynamic properties of this interaction, studied by calorimetric titrations of the heats of binding with this ligand, reveal a Kd of 12 +/- 2 microM at 25 degrees C and pH 7.4, a corresponding delta G of -6.7 +/- 0.1 kcal/mol, a delta H of -3.6 +/- 0.1 kcal/mol, and a delta S of 10.5 +/- 0.8 eu. The intrinsic fluorescence of r-K1 decreases by approximately 44% when its binding site is saturated with EACA, and titrations of this perturbation with EACA lead to calculation of a Kd of approximately 13 microM, a value in good agreement with that obtained from titration calorimetric analysis. EACA represents the strongest binding ligand of a variety of simple aliphatic omega-amino acids examined. A cyclic analogue of EACA, trans-4-(aminomethyl)cyclohexanecarboxylic acid, interacts with r-K1 with an approximate 12-fold tighter Kd (1.0 +/- 0.2 microM). Investigations by DSC, at pH 7.4, demonstrate that a significant stabilization of the r-K1 structure occurs when EACA binds to this domain. The temperature of maximum heat capacity change (Tm) in the thermal denaturation of r-K1 increases from approximately 340.8 to 359.1 K as a consequence of EACA binding. These studies demonstrate that a fully functional EACA-binding kringle from HPg can be expressed and secreted in E. coli, purified by techniques that do not require refolding, and investigated as an independent structural unit.

Peptides with more than one 106-amino acid sequence motif are needed to mimic the structural stability of spectrin.

The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the alpha-subunit and 17 in the beta-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180; Parry, D. A. D., Dixon, T. W., and Cohen, C. (1992) Biophys. J. 61, 858-867). In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation.

In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionic strength effect on the thermal unfolding of alpha-spectrin peptides.

In previous work, we have shown that the ionic strength-mediated differences found for the hydrodynamic dimensions of the human erythrocyte spectrin are not caused by secondary structural changes, but are caused more probably by subtle changes in tertiary interactions (LaBrake, C. C., Wang, L., Keiderling, T. A., and Fung, L. W.-M. (1993) Biochemistry 32, 10296-10302.). The substructure of spectrin has been suggested to be composed largely of triple alpha-helical bundle structural domains in tandem. In the present study, we used fluorescence and circular dichroism methods to study ionic strength effects on intact spectrin dimers and on recombinant peptides of spectrin domains of different lengths. We observed little ionic strength effect on the thermal unfolding temperature, Tm, values in these systems. However, we found that ionic strength-induced cooperativity in the unfolding processes was similar for the spectrin dimer and for peptides with two or three domains, as measured by entropy changes (DeltaSm). Although single-domain peptides exhibited rather variable DeltaSm values, depending on the specific domain, they showed little salt effects on the DeltaSm values themselves. This suggests that spectrin undergoes subtle ionic strength-induced conformational changes, probably near the interdomain regions of the molecule. These conformational changes may be responsible for the observed hydrodynamic and unfolding properties in intact spectrin under different ionic strength conditions. We suggest that recombinant peptides of various lengths may serve as models for studying the structural flexibility in spectrin.

Functional independence of the kringle 4 and kringle 5 regions of human plasminogen.

As part of continuing studies to evaluate whether the kringle domain regions of human plasminogen (HPg) exhibit independent conformational properties, simple model systems are required. Toward this end, we have constructed cDNA regions of HPg encoding its kringle 4 ([K4HPg]) and kringle 4-5 ([K4HPgK5HPg]) regions, expressed these gene fragments in bacterial cells, and purified the recombinant (r) products. The resulting r-[K4HPgK5HPg] was also employed to obtain the r-[K5HPg] domain of HPg by limited elastolytic digestion of this double-kringle polypeptide. The omega-amino acid ligand binding properties and thermal denaturation characteristics of r-[K4HPg], r-[K5HPg], and r-[K4HPgK5HPg] were determined, along with those for the [K5HPg] domain linked to the protease (P) region of HPg ([K5HPg]P). This allowed us to evaluate whether the conformational properties of the [K5HPg] module were influenced by the presence of its neighboring domains in HPg. The temperature midpoint of maximum heat capacity, Tm (and calorimetric enthalpy, delta H), for thermal denaturation of r-[K4HPg] was 57.8 degrees C (79.8 kcal/mol) in the absence of epsilon-aminocaproic acid (EACA) and 70.8 degrees C (93.7 kcal/mol) in the presence of that ligand. The corresponding values for isolated r-[K5HPg] were 50.4 degrees C (78.4 kcal/mol) and 61.0 degrees C (89.8 kcal/mol), respectively. These parameters for the isolated kringles were essentially unchanged when these same kringle domains were present in the r-[K4HPgK5HPg] and [K5HPg]P covalently linked pairs. Similarly, the thermodynamic characteristics (delta G, delta H, and delta S) that describe the binding energy of EACA to r-[K4HPg] at 25 degrees C were -6.3 kcal/mol, -4.5 kcal/mol, and 6.0 eu, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammaglobin is found in breast tissue as a complex with BU101.

The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.