Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coli lie in close proximity [Jung, K., Jung, H., Wu, J., Privé, G.G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. In this study, Glu269 was replaced with His in wild-type permease, leading to the presence of bis-His residues between helices VIII and X. Wild-type and Glu269-->His permease containing a biotin acceptor domain were purified by monomeric avidin affinity chromatography, and binding of Mn2+ was studied by electron paramagnetic resonance (EPR) spectroscopy. The amplitude of the Mn2+ EPR spectrum is reduced by the Glu269-->His mutant, while no change is observed in the presence of wild-type permease. The Glu269-->His mutant contains a single binding site for Mn2+ with a KD of about 43 microM, and Mn2+ binding is pH dependent with no binding at pH 5.0, stoichiometric binding at pH 7.5, and a midpoint at about pH 6.3. The results confirm the conclusion that helices VIII and X are closely opposed in the tertiary structure of lac permease and provide a novel approach for studying helix proximity, as well as solvent accessibility, in polytopic membrane proteins.     

Sound direction modifies the inhibitory as well as the excitatory frequency tuning characteristics of single neurons in the frog torus semicircularis (inferior colliculus)

We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragments through a 13 amino acid acceptor peptide translationally fused to the C-terminus of the Fd chain. The Fab-peptide fusion proteins were secreted to the periplasm of Escherichia coli, purified, and biotinylated in vitro using biotin ligase, biotin, and ATP. The E. coli biotin ligase (the BirA protein) was produced as a novel N-terminal fusion protein with glutathione S-transferase (GST) and purified in one step from bacterial cell lysate using a Glutathione Sepharose affinity column. The purified fusion protein worked as such (without cleavage of the GST part) for the in vitro biotinylation of the Fab fragments. After the removal of nonbiotinylated Fab fragments by monomeric avidin chromatography, the overall yield of biotinylated Fab was 40%. The site-specifically biotinylated Fab fragments (BioFab) were tested in streptavidin-coated microtitration wells, to which they were shown to bind linearly with respect to the amount of BioFab added, specifically as indicated by biotin inhibition, and tightly with a half-life of several days. Moreover, the enzymatic BioFab exhibited uniform antigen binding affinity unlike the same recombinant Fab fragments biotinylated through random chemical conjugation to surface lysines. Finally, the BioFab demonstrated its potential as a well-behaving immunoassay reagent in a model competitive assay for estradiol.     

Leukotriene receptors

The current challenge in research on leukotriene receptors is to clone these molecules. Traditional protein purification approaches have not been successful in providing sequence information. Solubilization of cys-LT1 has been achieved but results in the dissociation of G-proteins and the loss of high affinity binding (Mong et al., 1986b; Mong and Sarau, 1990), while cys-LT2 activity cannot be monitored by other than functional assays and there have not been any purification attempts. Partial purification of B-LT has been reported but has not been continued to homogeneity (Sherman et al., 1992; Votta et al., 1990; Miki et al., 1990). Nor have attempts to clone these receptors through either homology screening or expression cloning been successful. The cloning of the prostanoid receptors, described in detail elsewhere in this volume, has shown that these receptors belong to a distinct family within the G-protein-coupled receptor superfamily. It is probable, therefore, that the leukotriene receptors will also belong to a separate group within this superfamily since phylogenic comparisons have shown that receptors displaying high affinity for structurally related ligands exist as discrete families. Recently, a human cDNA encoding an orphan FMLP-related receptor cloned from HL60 cells of myeloid lineage was identified as the receptor for another eicosanoid, lipoxin A (Fiore et al., 1994). FMLP has a similar profile of biological actions to LTB4. Moreover, LTD4 showed a high degree of cross-reactivity with this receptor with an affinity only 20-fold less that of lipoxin A, although LTB4 was inactive. It remains to be determined whether the leukotriene receptors will fall into this class of receptors. The cloning of the leukotriene receptors will allow identification of the different receptor types and subtypes and potentially splice variants. Evaluation of currently developed antagonists at these receptor types could also open the way for novel therapies for inflammatory conditions.     

The impact of organizational factors on mental health professionals'' involvement with families

As an extension of previous study (de Vries et al., 1995, J. Biol. Chem., 270, 8712-8722) the acceptor specificity of recombinant FucT VI, expressed in insect cells as soluble enzyme, and purified from the growth medium by affinity chromatography, was analyzed toward a broad panel of oligosaccharide and glycoprotein substrates. It was found that FucT VI effectively utilizes any type-2-chain based structure (Gal beta 1-->4GlcNAc-R). Neutral as well as sialylated structures are fucosylated with high efficiency. To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. FucT VI had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3. Thus, FucT VI, although different from FucT III, IV, and V in acceptor properties, seems to bind the acceptor in a similar way.     

Molecular recognition in a post-translational modification of exceptional specificity. Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase

We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.     

Estimation of unit risk for coke oven emissions

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.     

Purification, regulation, and molecular and biochemical characterization of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain deltaH

We discovered that Methanobacterium thermoautotrophicum strain DeltaH possessed pyruvate carboxylase (PYC), and this biotin prototroph required exogenously supplied biotin to exhibit detectable amounts of PYC activity. The enzyme was highly labile and was stabilized by 10% inositol in buffers to an extent that allowed purification to homogeneity and characterization. The purified enzyme was absolutely dependent on ATP, Mg2+ (or Mn2+ or Co2+), pyruvate, and bicarbonate for activity; phosphoenolpyruvate could not replace pyruvate, and acetyl-CoA was not required. The enzyme was inhibited by ADP and alpha-ketoglutarate but not by aspartate or glutamate. ATP was inhibitory at high concentrations. The enzyme, unlike other PYCs, exhibited nonlinear kinetics with respect to bicarbonate and was inhibited by excess Mg2+, Mn2+, or Co2+. The 540-kDa enzyme of A4B4 composition contained a non-biotinylated 52-kDa subunit (PYCA) and a 75-kDa biotinylated subunit (PYCB). The pycB gene was probably monocistronic and followed by a putative gene of a DNA-binding protein on the opposite strand. The pycA was about 727 kilobase pairs away from pycB on the chromosome and was probably co-transcribed with the biotin ligase gene (birA). PYCA and PYCB showed substantial sequence identities (33-62%) to, respectively, the biotin carboxylase and biotin carboxyl carrier + carboxyltransferase domains or subunits of known biotin-dependent carboxylases/decarboxylases. We discovered that PYCB and probably the equivalent domains or subunits of all biotin-dependent carboxylases harbored the serine/threonine dehydratase types of pyridoxal-phosphate attachment site. Our results and the existence of an alternative oxaloacetate synthesizing enzyme phosphoenolpyruvate carboxylase in M. thermoautotrophicum strain DeltaH (Kenealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett. 14, 7-10) raise several questions for future investigations.     

Cysteine scanning mutagenesis of helix V in the lactose permease of Escherichia coli

Using a functional lactose permease mutant devoid of Cys (C-less permease), each amino acid residue in putative transmembrane helix V was replaced individually with Cys (from Met145 to Thr163). Of the 19 mutants, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-less permease). Cys replacement of Gly147 or Trp151 essentially inactivates the permease (< 10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030] demonstrate that neither of these residues is important for activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to C-less permease with the exception of Trp151-->Cys and single Cys154 permeases which are present in reduced amounts. Finally, only three of the single-Cys mutants are inactivated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.     

Inhibition of tumor cell invasion through matrigel by a peptide derived from the domain II region in urinary trypsin inhibition

Urinary trypsin inhibitor (UTI) has a multipotent inhibitory effect on proteases such as trypsin, chymotrypsin, plasmin, human leukocyte elastase, or hyaluronidase. UTI can bind easily to its receptors on various types of tumor cells (human ovarian cancer HOC-I cells, human choriocarcinoma SMT-cc1 cells, and murine Lewis lung carcinoma 3LL cells). Our results show that the UTI receptors of some tumor cells have a possible role in modulating plasmin activity on the cell surface and prevention of tumor cell invasion and metastasis (H. Kobayashi et al., J. Biol. Chem., 269; 20642-20647, 1994). UTI interacts with tumor cells as a negative modulator of the invasive cells. We investigated whether this effect may be mediated by UTI binding to the cell surface receptors. In addition, the role of peptide sequences from each UTI domain and their interaction with tumor cells were investigated. UTI derivatized with biotin or FITC was taken up by tumor cells in a dose-dependent manner. This cell association was inhibited with a monoclonal antibody D1, which specifically recognizes NH2 terminus (domain I) of UTI. The binding was inhibited by fluid phase UTI, but not HI-8, COOH terminus (domain II) of UTI, suggesting that UTI binds to cells through a site in the UTI domain I. Furthermore, we found that UTI, HI-8 and a number of peptides containing Arg-Gly-Pro-Cys-Arg-Ala-Phe-Ile promoted the inhibition of tumor cell invasion. This site corresponds to the plasmin-inhibiting domain within HI-8. The possibility that UTI binding to tumor cells might be involved in the prevention of tumor cell invasion in vitro was excluded since HI-8, lacking domain I, promotes the inhibition of tumor cell invasion with essentially the same affinity as UTI. All these data allow us to conclude that inhibition of tumor cell invasion is mediated by domain II, which possesses anti-plasmin activity.     

Entropy-driven intermediate steps of oxygenation may regulate the allosteric behavior of hemoglobin

When the oxygen binding isotherms of human, bovine and fallow deer (Dama-Dama) hemoglobins are measured at different temperatures either by optical or calorimetric techniques, analyses according to the Adair's formalism show that at least one of the intermediate steps of ligation has a positive enthalpy change, i.e., absorbs rather than emitting heat, indicating that it is entropy rather than enthalpy driven (Bucci, E., et al. 1991. Biochemistry. 30:3195-3199; Bucci, E., et al. 1993. Biochemistry. 32:3519-3526; Johnson, C., et al. 1992. Biochemistry. 31:10074-10082; Johnson, C., et al. 1995. Biophys. Chem. 59:107-117). This phenomenon is confirmed in systems in which the beta82 lysines of human hemoglobin are covalently cross-linked by acylation with dicarboxylic acids of increasing length, namely the fumaryl (four-carbon-long), adipoyl (six-carbon-long), and sebacoyl (10-carbon-long) residues. Consistently in all of the systems here reported, the enthalpy excursions are masked by compensatory entropy changes, which keep the free energy of ligand binding constant for the first three steps of oxygenation. Furthermore, the cooperativity index and the overall oxygen affinity seem to be correlated to the positive enthalpy excursions of the intermediate steps of ligation. Fumaryl-Hb (hemoglobin cross-linked with a fumaryl residue, four carbons) with the lowest absorption of heat has the highest affinity and lowest cooperativity index. Adipoyl-Hb (hemoglobin cross-linked with an adipoyl residue, six carbons) has the highest absorption of heat and the highest cooperativity index. It appears that nonuniform heat release by the intermediates of oxygenation is part of the allosteric phenomena in hemoglobin systems. There is not enough information that would allow assigning these phenomena to the interplay of the various conformations described for hemoglobin besides the classic T (Fermi et al. 1984. J. Mol. Biol. 175:159-174) and R (Shanaan. 1983. J. Mol. Biol. 171:31-59), as listed at the end of the Discussion. The possibility cannot be excluded that entropy-driven steps characterize new conformational transitions still to be described.     

Purification and polypeptide composition of Semliki Forest virus RNA polymerase

A purification method for Semliki Forest virus-specified RNA-dependent RNA polymerase from BHK cells is described. The procedure entails (i) the preparation of a crude cell lysate by Dounce homogenization of cells 3-5 h post-infection, (ii) differential centrifugation to give a 15 000 g 'mitochondrial' pellet, (iii) equilibrium centrifugation on discontinuous sucrose gradients (Friedman et al. 1972) to give a membranous band of density 1-16 g/ml, (iv) solubilization with Triton N-101 and velocity centrifugation to give a 25S solubilized polymerase complex and (v) affinity chromatography through an oligo (dT)-cellulose matrix bearing immobilized 42S virus particle RNA. The overall purification was approx. 360-fold with a 5% recovery of activity. Of the various intermediate fractions in the purfication procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNAs found in infected cells. Other fractions incorporated precursor only into replicative intermediate (RI) or replicative from (RF). Analysis of the product RF showed that it was of the same size and could bind to the same extent to oligo (dT)-cellulose as the RF isolated directly from lysates of infected cells. Displacement hybridization and ribonuclease digestion suggested that the purified polymerase could only complete previously initiated progeny positive strands using negative strands as template and, even in its most highly purified form, was still tightly bound to its template. Analysis on polyacrylamide slab gels revealed the presence of three 35S-labelled polypeptides in the purified polymerase preparation, but a polypeptide which had identical electrophoretic mobility to the lowest mol. wt. polypeptide of the purified polymerase was also present in material from mock-fected cells which had been taken through the purification procedure. From these results we conclude that only two virus-specified polypeptides are present in the polymerase. A scheme for the synthesis of these polypeptides is presented in the accompanying paper.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin-Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by alpha-thrombin (IC50 = 28 microg/ml). A single band of approximately 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation (Ic50 = 0.19 microg/ml) compared to bothrojaracin (IC50 = 0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

Functional analysis of GnRH receptor ligand binding using biotinylated GnRH derivatives

The objective of this study was to determine whether the gonadotrophin-releasing hormone (GnRH) ligand binds to the GnRH receptor (GnRH-R) with either the N- and C-termini or the beta-II turn pointing towards the cell. The functionality of GnRH and two biotinylated GnRH derivatives, biotin [D-Lys6]GnRH and biotin [Gln1]GnRH biotinylated at positions 6 and 1, respectively was assessed. Streptavidin was also used in combination with these peptides to investigate the effects of the steric hindrance caused by this molecule on ligand binding when bound to the biotin molecules at the two positions. GnRH bound to the receptor with high affinity, which was not affected by the addition of streptavidin. Both the biotinylated derivatives bound to the receptor though with lower affinities than GnRH. The biotin [D-Lys6]GnRH-streptavidin complex bound to the receptor albeit with lower affinity compared to biotin [D-Lys6]GnRH only, although it maintained its ability to cause receptor internalisation. The ability of the biotin [Gln1]GnRH to bind to the receptor was abolished in the presence of excess streptavidin. Both GnRH and biotin [D-Lys6]GnRH stimulated total inositol phosphate production whereas biotin [Gln1]GnRH exhibited GnRH antagonist activity. It appears that the small biotin molecule can be accommodated within the binding pore when attached to position 1 of the ligand but not when complexed to streptavidin. The fact that biotin [D-Lys6]GnRH maintains functionality when complexed to streptavidin while biotin [Gln1]GnRH does not, suggests that the N- and possibly the C-termini are required for receptor binding. Thus the most likely binding orientation for the ligand is with the N- and C-termini pointing inwards with the residue at position 6 pointing away from the binding site.     

Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains

The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and purification of diphosphoinositol pentakisphosphate kinase, which synthesizes the inositol pyrophosphate bis(diphospho)inositol tetrakisphosphate

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) were recently identified as inositol phosphates which possess pyrophosphate bonds. The molecular mechanisms that regulate the cellular levels of these compounds are not yet characterized. To pursue this question, we have previously purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants [Voglmaier, S. M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4305-4310]. We now report the identification and purification of another novel kinase, diphosphoinositol pentakisphosphate (PP-IP5) kinase, which uses PP-IP5 as a substrate to form bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) in soluble fractions of rat forebrain. The purified protein, a monomer of 56 kDa, displays high affinity (Km = 0.7 microM) and selectivity for PP-IP5 as a substrate. The purified enzyme also can transfer a phosphate from bis-PP-IP4 to ADP to form ATP. This ATP synthase activity is an indication of the high phosphoryl group transfer potential of bis-PP-IP4 and may represent a physiological role for PP-IP5 and bis-PP-IP4.     

Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique

The Serial Analysis of Gene Expression (SAGE) method, described in 1995 by Velculescu et al ., represents a powerful means to compare gene expression between two mRNA populations. An improvement to SAGE that removes contaminating linker molecules, which compromise the efficiency of the method, has been developed. This modification utilises biotinylated PCR primers, which generate biotinylated linkers at an early stage in the SAGE protocol, thus allowing removal of the unwanted linkers by binding to streptavidin-coated magnetic beads at a later stage. The application of this modification resulted in the rapid generation of high ditag yields and clones with large average insert sizes.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

Binding of monoclonal antibody 4B1 to homologs of the lactose permease of Escherichia coli

The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+ translocation in the lactose permease of Escherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996, Biochemistry 35;990-998). Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major Facilitator Superfamily (MFS), which includes five homologous oligosaccharide/H+ symporters, reveals interesting variations. 4B1 binds to the Citrobacter freundii lactose permease or E. coli raffinose permease with resultant inhibition of transport activity. Because E. coli raffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide backbone at this position. Consistently, E. coli lactose permease with Pro in place of Gly254 also binds 4B1. In contrast, 4B1 binding is not observed with either Klebsiella pneumoniae lactose permease or E. coli sucrose permease. When the epitope is transferred from E. coli lactose permease (residues 245-259) to the sucrose permease, the modified protein binds 4B1, but the mAb has no significant effect on sucrose transport. The studies provide further evidence that the 4B1 epitope is restricted to loop VII/VIII, and that 4B1 binding induces a highly specific conformational change that uncouples substrate and H+ translocation.     

Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells

Recently, O'Reilly et al. (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277-285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simple in vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 microg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos.     

Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.