Separation of the nativelike intermediate from unfolded forms during refolding of ribonuclease A

In an effort to determine structural properties of the nativelike intermediate (i.e., IN) which forms during the refolding of RNase A, refolding samples were subjected to rapid HPLC gel filtration which allowed us to separate IN from unfolded forms of RNase. The comparison of these samples, enriched in IN and depleted of unfolded forms, with unseparated control samples at the same stage of refolding allowed certain conclusions to be drawn concerning the properties of IN. First, the results show that the transition from IN to native RNase occurs with only small changes in fluorescence. This means that the major fluorescence changes seen during normal refolding experiments must be associated with changes in proline isomerization of unfolded species and/or with the refolding step itself but not with the IN----N step. Second, the fluorescence assay for isomerization of proline-93 shows that IN exists with proline-93 in a state of isomerization identical with or very similar to native RNase; i.e., proline-93 is cis in IN and not trans as suggested by others. All results are semiquantitatively consistent with our earlier refolding model and not nearly so consistent with alternative models which assume that most or all of the slow-refolding forms of RNase have proline-93 in the incorrect trans state.     

Phase metastability and supercooled metastable state of diundecanoylphosphatidylethanolamine bilayers

Aqueous dispersons of L-alpha-phosphatidylethanolamine (PE) with identical saturated acyl chains are known to exhibit gel-state metastability. It is also known that the metastability in PE becomes more pronounced with decreasing acyl chain-length. In an attempt to study the metastable phase behavior of PE, we have synthesized diundecanoylphosphatidylethanolamine (diC11PE) and examined its polymorphic phase behavior. A single endothermic transition at 38 degrees C is detected between 10 and 55 degrees C by DSC for the nonheated sample of diC11PE in excess water. An immediate second heating scan done after cooling slowly of the same sample from the liquid-crystalline state shows a smaller endothermic transition at a lower temperature, 18 degrees C. However, the high-temperature transition at 38 degrees C can be detected, if the sample which has been heated above 38 degrees C is quench cooled from the liquid-crystalline to a temperature between 18 and 38 degrees C. Furthermore, two endothermic transitions at 18 and 38 degrees C and an exothermic transition at 19 degrees C are recorded for diC11PE after quench supercooling of the sample from the liquid-crystalline state to an appropriate temperature below 10 degrees C. The gel-state metastability of diC11PE can be most appropriately explained in terms of changes in interbilayer headgroup-headgroup interactions. It is suggested that the kinetically trapped supercooled metastable state may be a multilamellar structure with melted acyl chains but with strong interbilayer headgroup-headgroup interactions.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.     

Isolation and interrelationships of the multiple molecular tissue-type and urokinase-type plasminogen activator forms produced by cultured human umbilical vein endothelial cells

Primary and early subcultures (1st- to 3rd passage) of human umbilical vein endothelial cells produce tissue-type plasminogen activator (t-PA) antigen, consisting only of a major Mr 110,000 t-PA form. Later subcultures (greater than 4th passage) produce increasing amounts of t-PA antigen, consisting of a major Mr 110,000 and a minor Mr 68,000 form as well as increasing amounts of urokinase-type plasminogen activator (u-PA) antigen, consisting of a minor Mr 95,000 and major Mr 54,000 form. All of the major plasminogen activator forms were purified to homogeneity from 72 h serum-free conditioned media (3 liters, 1-1.8 x 10(9) cells) by a combination of immunoaffinity and gel filtration chromatography. Typically, 4th to 6th passage cultures produced/secreted t-PA-type proteins consisting of an inactive Mr 110,000 (220 IU/mg) and active Mr 68,000 (76,500 IU/mg) form representing about 39 and 8%, respectively, of the total starting sodium dodecyl sulfate stable t-PA activity, and u-PA-type proteins consisting of an inactive Mr 95,000 (700 IU/mg) and active Mr 54,000 (81,000 IU/mg) form representing about 9 and 38%, respectively, of the total starting sodium dodecyl sulfate stable u-PA activity. The isolated Mr 68,000 t-PA and Mr 54,000 u-PA proteins, exist only as two-chain forms in the absence of aprotinin and as mixtures of single- and two-chain proteins in the presence of aprotinin. Treatment with nucleophilic agents completely dissociated the Mr 110,000 t-PA and Mr 95,000 u-PA proteins into their respective Mr 68,000 t-PA and Mr 54,000 u-PA activity forms and a common Mr 46,000 protein, confirming the enzyme-inhibitor complex nature of these inactive plasminogen activator forms.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.     

The cytotoxins alpha-sarcin and ricin retain their specificity when tested on a synthetic oligoribonucleotide (35-mer) that mimics a region of 28 S ribosomal ribonucleic acid

An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo.     

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).     

A high-resolution C-banding technique

A high-resolution C-banding technique is described. The major advantages of this method are its simplicity and reliability. Because this technique allows the recognition of unusual C-band heteromorphisms, it can be very useful in population cytogenetic studies.