Does the fluorescence quencher acrylamide bind to proteins?

We have studied the protein concentration dependence of the acrylamide quenching of the fluorescence of the proteins, human serum albumin and monellin, and we have found no such dependence for the concentration range of 0.5-20 mg/ml. These quenching studies were performed by fluorescence lifetime measurements using phase/modulation fluorometry. We have also performed equilibrium dialysis studies, which show no large degree of association of acrylamide with serum albumin, and we have found that acrylamide has only a small effect on the activity of selected enzymes. These various studies do not indicate the existence of strong acrylamide-protein interactions and are in discord with a recent report by Blatt et al. in this journal (Blatt, E., Husain, A. and Sawyer, W.H. (1986) Biochim. Biophys. Acta 871, 6-13).     

The complete amino-acid sequence of the bilin-binding protein from Pieris brassicae and its similarity to a family of serum transport proteins like the retinol-binding proteins

The amino-acid sequence from the bilin binding protein (BBP) of the butterfly Pieris brassicae has been determined. The apoprotein with a length of 173 amino-acid residues has a molecular mass of 19,676 Da. The sequence analysis was performed by automated Edman degradation of the intact apoprotein and of fragments as large as possible generated from different digestions. The 3-dimensional structure of BBP, determined by Huber et al. (Huber, R., Schneider, M., Epp, O., Mayr, I., Messerschmidt, A., Pflugrath, J. & Kayser, H. (1987) J. Mol. Biol. 195, 423-434 and Huber, R., Schneider, M., Mayr, I., Müller, R., Deutzmann, R., Suter, F., Zuber, H., Falk, H. & Kayser, H. (1987) J. Mol. Biol. 198, 499-513) down to 2-A resolution, exhibits a similar conformation to the human retinol binding protein. Sawyer (Sawyer, L. (1987) Nature (London) 327, 659) demonstrated that proteins from a wide variety of sources can be gathered into a "superfamily". Computer searches of data banks yielded in a new member of this superfamily, namely human alpha 1-acid glycoprotein. One of the functions of the listed proteins is to bind and transport small hydrophobic molecules in serum.     

Comparison between two classes of selective EP(3) antagonists and their biological activities

Two different series of very potent and selective EP(3) antagonists have been reported: a novel series of ortho-substituted cinnamic acids [Belley, M., Gallant, M., Roy, B., Houde, K., Lachance, N., Labelle, M., Trimble, L., Chauret, N., Li, C., Sawyer, N., Tremblay, N., Lamontagne, S., Carrière, M.-C., Denis, D., Greig, G. M., Slipetz, D., Metters, K. M., Gordon, R., Chan, C. C., Zamboni, R. J. Bioorg. Med. Chem. Lett.2005, 15, 527] and the acylsulfonamides of ortho-(arylmethyl)cinnamates. [(a) Juteau, H., Gareau, Y., Labelle, M., Sturino, C. F., Sawyer, N., Tremblay, N., Lamontagne, S., Carrière, M.-C., Denis, D., Metters, K. M. Bioorg. Med. Chem. 2001, 9, 1977; (b) Juteau, H., Gareau, Y., Labelle, M., Lamontagne, S., Tremblay, N., Carrière, M.-C., Denis, D., Sawyer, N., Metters, K. M. Bioorg. Med. Chem. Lett.2001, 11, 747] The structural differences between the two series, along with their biological activity in vivo, in vitro, and metabolism, are analyzed. Some of those compounds, including hybrids containing the best structural features of both series, possess K(i) as low as 0.6 nM on the EP(3) receptor.     

Isolation and characterization of a tropomyosin binding protein from human blood platelets

We have isolated a tropomyosin binding protein (TMBP) from human platelets using isoelectric fractionation, hydroxylapatite chromatography, and affinity chromatography on skeletal muscle tropomyosin-Affi-Gel 15. TMBP is a 67,000-Da monomeric protein that binds to muscle and nonmuscle tropomyosin affinity resins. Its affinity for platelet tropomyosin is greater than for rabbit skeletal or chicken gizzard tropomyosin, and greater than that of troponin for all tropomyosin affinity resins tested. TMBP forms a complex with platelet tropomyosin that can be isolated on G-150. The approximate molar stoichiometry is 1:1. Troponin and TMBP have distinct binding sites on skeletal tropomyosin since binding of TMBP to tropomyosin-Affi-Gel 15 is not affected by previous saturation of the column with troponin (or vice versa). The amino acid composition of TMBP is virtually identical with that of human serum albumin, and is similar to those of beta-actinin (Heizmann, C. W., Müller, G., Jenny, E., Wilson, K. J., Landon, F., and Olomucki, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 74-77) and acumentin (Southwick, F. S., and Stossel, T. P. (1981) J. Biol. Chem. 256, 3030-3036). The protein we have isolated is the first nonmuscle protein other than actin that has been shown to bind to tropomyosin. Results in an accompanying paper show that this tropomyosin binding protein is identical with human serum albumin (Hitchcock-DeGregori, S. E., Gerhard, M. D., and Brown, W. E. (1985) J. Biol. Chem. 260, 3228-3231).     

Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.     

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.     

Resonance energy transfer between cysteine-34, tryptophan-214, and tyrosine-411 of human serum albumin

Reaction of p-nitrophenyl anthranilate with human serum albumin at pH 8.0 results in esterification of a single anthraniloyl moiety with the hydroxyl group of tyrosine-411. The absorption spectrum of the anthraniloyl group overlaps the fluorescence emission of the single tryptophan residue at position 214. This study complements that of the preceding paper [Suzukida, M., Le, H. P., Shahid, F., McPherson, R. A., Birnbaum, E.R., & Darnall, D. W. (1983) Biochemistry (preceding paper in this issue)] where an azomercurial group was introduced at cysteine-34. Anthraniloyl fluorescence was also quenched by the azomercurial absorption at Cys-34. Thus measurement of resonance energy transfer between these three sites allowed distances to be measured between Cys-34 in domain I, Trp-214 in domain II, and Tyr-411 in domain III of human serum albumin. At pH 7.4 in 0.1 M phosphate the Trp-214 leads to Tyr-411, Tyr-411 leads to Cys-34, and Trp-214 leads to Cys-34 distances were found to be 25.2 +/- 0.6, 25.2 +/- 2.1, and 31.8 +/- 0.8 A, respectively.     

Novel regulatory enhancer in the nuclear gene of the human mitochondrial ATP synthase beta-subunit

The system coordinating expressions of nuclear coded mitochondrial proteins was investigated by examination of the 5'-flanking region of the human mitochondrial ATP synthase beta-subunit gene. The promoter activity was measured by a transient expression of a chloramphenicol acetyltransferase (CAT) gene connected with various 5'-deletion mutants of the 5'-flanking region. In this experiment, at least two regions enhanced this promoter activity and at least one region repressed it. In one of the enhancing regions, a consensus sequence was found for the genes of other mitochondrial proteins such as those for cytochrome c1 (Suzuki, H., Hosokawa, Y., Nishikimi, M., and Ozawa, T. (1989) J. Biol. Chem. 264, 1368-1374) and the pyruvate dehydrogenase alpha-subunit (Maragos, C., Hutchison, W. M., Hayasaka, K., Brown, G. K., and Dahl, H.-H. M. (1989) J. Biol. Chem. 264, 12294-12298; Ohta, S., Endo, H., Matsuda, K., and Kagawa, Y. (1989) Ann. N. Y. Acad. Sci. 573, 458-460). The characteristics of this enhancing element were examined by introducing a synthetic oligonucleotide element into the CAT plasmid with a deleted enhancing element. The resulting plasmid showed full recovery of promoter activity, and this activity was independent of the orientation or location of the insert. Therefore, this is an enhancer that may be common to the nuclear genes of some mitochondrial proteins involved in energy transduction.     

Amelogenin interacts with cytokeratin-5 in ameloblasts during enamel growth

The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 - 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

A common diagnostic feature of glycosylinositol phospholipid (GPI)-anchored proteins is their release from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). However, some GPI-anchored proteins are resistant to this enzyme. The best characterized example of this subclass is the human erythrocyte acetylcholinesterase, where the structural basis of PI-PLC resistance has been shown to be the acylation of an inositol hydroxyl group(s) (Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T. L. (1988) J. Biol. Chem. 263, 18766-18775). Both PI-PLC-sensitive and resistant GPI-anchor precursors (P2 and P3, respectively) have been found in Trypanosoma brucei, where the major surface glycoprotein is anchored by a PI-PLC-sensitive glycolipid anchor. The accompanying paper (Mayor, S., Menon, A. K., Cross, G. A. M., Ferguson, M. A. J., Dwek, R. A., and Rademacher, T. W. (1990) J. Biol. Chem. 265, 6164-6173) shows that P2 and P3 have identical glycans, indistinguishable from the common core glycan found on all the characterized GPI protein anchors. This paper shows that the single difference between P2 and P3, and the basis for the PI-PLC insusceptibility of P3, is a fatty acid, ester-linked to the inositol residue in P3. The inositol-linked fatty acid can be removed by treatment with mild base to restore PI-PLC sensitivity. Biosynthetic labeling experiments with [3H]palmitic acid and [3H]myristic acid show that [3H]palmitic acid specifically labels the inositol residue in P3 while [3H]myristic acid labels the diacylglycerol portion. Possible models to account for the simultaneous presence of PI-PLC-resistant and sensitive glycolipids are discussed in the context of available information on the biosynthesis of GPI-anchors.     

Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine

We have previously discovered the opium alkaloid noscapine as a microtubule interacting agent that binds to tubulin, alters the dynamics of microtubule assembly, and arrests mammalian cells at mitosis (Ye, K., Ke, Y., Keshava, N., Shanks, J., Kapp, J. A., Tekmal, R. R., Petros, J., and Joshi, H. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1601-1606; Ye, K., Zhou, J., Landen, J. W., Bradbury, E. M., and Joshi, H. C. (2001) J. Biol. Chem. 276, 46697-46700; Zhou, J., Panda, D., Landen, J. W., Wilson, L., and Joshi, H. C. (2002) J. Biol. Chem. 277, 17200-17208). Here we show that noscapine does not compete with paclitaxel for tubulin binding and can efficiently inhibit the proliferation of both paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells (i.e. the parental cell line 1A9 and two derivative cell lines, 1A9PTX10 and 1A9PTX22, which harbor beta-tubulin mutations that impair paclitaxel-tubulin interaction (Giannakakou, P., Sackett, D. L., Kang, Y. K., Zhan, Z., Buters, J. T., Fojo, T., and Poruchynsky, M. S. (1997) J. Biol. Chem. 272, 17118-17125). Strikingly, these cells undergo apoptotic death upon noscapine treatment, accompanied by activation of the c-Jun NH(2)-terminal kinases (JNK). Furthermore, inhibition of JNK activity by treatment with antisense oligonucleotide or transfection with dominant-negative JNK blocks noscapine-induced apoptosis. These findings thus indicate a great potential for noscapine in the treatment of paclitaxel-resistant human cancers. In addition, our results suggest that the JNK pathway plays an essential role in microtubule inhibitor-induced apoptosis.     

A dynamic model for bilirubin binding to human serum albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.     

Further studies on human cholesterol-binding pancreatic protease/elastase 1. Immunological detection of analogous enzymes in several animal species and identification of the porcine-derived enzyme as protease E

Antibodies against the human cholesterol-binding pancreatic protease/elastase 1 (Sziegoleit, A., Linder, D., Schlüter, M., Ogawa, M., Nishibe, S. & Fujimoto, K. 1985) Eur. J. Biochem. 151, 595-599) recognize a distinct protein in the pancreas homogenate of various animal species. The CBPP/elastase 1-related porcine protease was purified and characterized. Its properties, including specificity, proved to be the same as those of the well classified porcine pancreatic protease E (Kobayashi, R., Kobayashi, Y. & Hirs, C.H.W. (1981) J. Biol. Chem. 256, 2460-2465). While the common features of all these proteins seem to be the proteolysis with elastase-like specificities (investigated for the human-, porcine-, dog- and rat-derived protein) and an isoelectric point at about pH 5 (determined for the pancreatic proteins from man, swine, rat dog and cattle), the bile salt and cholesterol-binding capacity varies significantly among the animal species. Charge shift crossed immunoelectrophoresis of the pancreatic proteins from rat, dog, cat, swine, horse, zebra, cattle and rabbit reveals that only the protein from rat pancreas binds the negatively charged bile salt sodium deoxycholate to an extent comparable to that of human CBPP/elastase 1. Thus, within the diverse elastase family, there seems to be a distinct enzyme which merits distinct classification.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

Kinetics of hemoprotein reduction and interprotein heme transfer

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W.T., Liem, H.H., Sutor, R.P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R.F., Gibbs, E.J., Hoeflin, E., Kosar, W.P., Kubera, G., Skowronek, C. A., Wong, N.M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (HmIII., ferriprotoporphyrin IX) was reduced to HmII (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of HmIII to HmII were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K congruent to 10(8)), and with hemopexin (HHx), which binds heme equimolarly (K congruent to 10(13)). The rate of reduction of HmIII to HmII on HSA was first order over several half-lives and linearly dependent on [S2O4(2-)]1/2. At [HSA]0/[HmIII] = 3, the kobsd was (5 X 10(-3) + 0.75[S2O4(2-)]1/2, and with [HSA]/[HmIII] approximately 25, the kobsd was (2 X 10(-3)) + 0.25[S2O4(2-)]1/2. The reduction of HmIII to HmII on human hemopexin (HHx) is much more rapid with kobsd = (2.5 X 10(3))[S2O4(2-)]1/2.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism by which dicyclohexylcarbodiimide and quinine inhibit K+ transport in rat liver mitochondria

Passive uptake of potassium acetate into the mitochondrial matrix can be induced by nigericin, a K+/H+ antiporter, or by A23187, a Mg2+/2H+ antiporter. The latter process is thought to reflect operation of the Mg2+-dependent, endogenous K+/H+ antiporter, but there is ambiguity with respect to the mechanism of K+ transport in this assay (Nakashima, R.A., and Garlid, K.D. (1982) J. Biol. Chem. 257, 9252-9254). Kinetic analysis of potassium acetate transport provides verification that Mg2+ depletion 1) unmasks the K+/H+ antiporter, 2) opens up an intrinsic anion uniporter, 3) has no effect on acetic acid transport, and 4) does not induce high K+ uniport conductance. Mg2+-dependent uptake of potassium acetate is thereby shown to be mediated specifically by operation of the endogenous K+/H+ antiporter, as previously proposed. An extension of this analysis confirms that N,N'-dicyclohexylcarbodiimide and quinine block potassium acetate uptake via specific action on the K+/H+ antiporter. These findings support those of a previous study (Martin, W.H., Beavis, A.D., and Garlid, K.D. (1984) J. Biol. Chem. 259, 2062-2065) in which binding of [14C]N,N'-dicyclohexylcarbodiimide to membrane proteins under selective conditions was used to identify an 82,000-dalton band as the protein responsible for K+/H+ antiport in mitochondria.     

Platelet tropomyosin binding protein is identical with serum albumin

We have shown that the platelet tropomyosin binding protein described in the accompanying paper (Gerhard, M. D., DiGirolamo, P. M., and Hitchcock-DeGregori, S. E. (1985) J. Biol. Chem. 260, 3221-3227) is identical with human serum albumin. The immunological determinants are completely shared; the tryptic peptide maps are the same; the proteins comigrate on two-dimensional gels; and the amino acid sequences of the first 33 amino acids are the same. Although human serum albumin in plasma or commercially prepared protein will not bind tropomyosin-Affi-Gel 15, it will bind following purification from plasma by chromatography on hydroxylapatite.     

Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.     

Isolation and properties of the active gamma subunit of phosphorylase kinase

A catalytically active gamma subunit of phosphorylase kinase was prepared from pure, but inactive, gamma subunit obtained by reverse-phase high pressure liquid chromatography (HPLC). The HPLC procedure (Crabb, J. W., and Heilmeyer, L. M. J., Jr. (1984) J. Biol. Chem. 259, 6346-6350) leaves the isolated gamma subunit in 50% acetonitrile and 0.09% trifluoroacetic acid (pH 2.5) and assay of this species at pH 8.2 indicates that it is inactive. Reactivation occurred, however, when the HPLC-isolated gamma subunit was diluted into an ice-cold, pH 8.2 buffer containing both calcium and calmodulin. Optimum reactivation depended on time, temperature, concentration of the HPLC solvent components, gamma subunit concentration, pH, the presence of both calcium and calmodulin, and an additional protein such as bovine serum albumin or phosphorylase b. Studies of the reactivated gamma subunit in the presence of the reactivation mixture indicate that it may be equivalent to a gamma delta subunit complex previously isolated (Chan, K.-F. J., and Graves, D. J. (1982) J. Biol. Chem. 257, 5939-5947). Like the gamma delta subunit complex, the catalytic activity of the reactivated gamma subunit species is not significantly affected by pH within the range of pH 6.8-8.2 and is inhibited 70% by removal of Ca2+. A reactivated gamma subunit free of calmodulin was also obtained. This was done by first substituting agarose-bound calmodulin for free calmodulin in the reactivation procedure described above and, then, elution of the gamma subunit from the calmodulin-agarose with a solution containing 1.0 M Tris-Cl (pH 7.0), 1% Triton X-100, 1 mM EGTA, and 5 mM dithiothreitol. The activity of the isolated, active gamma subunit is insensitive to Ca2+ and is stimulated 1.4-fold in a calcium-dependent manner by the addition of calmodulin.