Hugo Fasold

Purification and characterization of a proteolytic enzyme from Candida albicans

Abstract In media containing proteins as the nitrogen source some strains of Candida albicans excrete proteolytic enzymes with an optimum of activity at pH 3.2. A 140-fold purification of the enzyme from one of these strains was achieved by gel filtration and chromatography on DEAE-Sephadex. The molecular weight of the protein was found to be 40 000; the frictional ratio, 1.3. The proteolytic activity was not influenced by the addition of heavy metal ions, SH-blocking reagents, p-bromophenacyl bromide, N-diazoacetylnorleucine methyl ester, or various inorganic ions. Cleavage of oxidized insulin β-chain and albumin demonstrated a low side-chain specificity; a preferential attack, on hydrophobic amino acid residues was observed after incubation for short periods of time.

Characterization and chemical modification of the Na+-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.

Intracellular localization of the 90 kDA heat shock protein (HSP90α) determined by expression of a EGFP–HSP90α-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90α fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP—Hsp90α was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP—Hsp90α could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP—Hsp90α are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP—Hsp90α with the nuclear membrane became visible.

The use of azidoarylimidoesters in RNA-protein cross-linking studies with Escherichia coli ribosomes

Abstract A series of related hetero-bifunctional RNA-protein cross-linking reagents has been prepared, carrying an imidoester or N -hydroxysuccinimide ester function at one end of the molecule, and a phenylazido function at the other. These compounds have been applied to RNA-protein cross-linking studies with ribosomal subunits, and one of them, p -azido-phenylacetic imidoester, has proved to be a particularly useful reagent for this purpose. The reagent first reacts specifically with protein amino groups, and subsequent photolysis of the azide group leads to cross-linking to the RNA in yields of up to 8% of the total protein. The whole reaction takes place under very mild conditions in aqueous solution. The individual proteins concerned in the cross-links have been identified by two-dimensional gel electrophoresis, and the existence of a covalent cross-link was confirmed by the isolation by two different methods of protein-oligonucleotide complexes carrying a 32 P label. Although most of the ribosomal proteins could be cross-linked to their corresponding ribosomal RNA within the individual subunits, RNA-protein cross-links at the ribosomal subunit interface were only detectable in vanishingly small amounts. The advantages of this type of genuine hetero-bifunctional reagent in RNA-protein cross-linking studies are discussed.

A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits.

Ribosomal subunits are assembled in the nucleus, and mature 40 S and 60 S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60 S ribosomal subunits involves the use of resealed nuclear envelopes. The export of ribosomal subunits from resealed nuclear envelopes is enhanced by cytoplasmic proteins. Here we present evidence that the export-promoting activity was due to the cytoplasmic 90 kDa heat-shock protein (Hsp90). Isolated, purified Hsp90 vastly enhanced the export of 60 S ribosomal subunits from resealed nuclear envelopes, while inhibition of Hsp90 function, either with the Hsp90-binding drug geldanamycin or with anti-Hsp90 antibodies, resulted in reduced release of 60 S ribosomal subunits. To confirm these findings under in vivo conditions, corresponding experiments were performed with Xenopus oocytes using microinjection techniques; the results obtained confirmed the findings obtained with resealed nuclear envelopes. These findings suggest that Hsp90 facilitates the nuclear export of 60 S ribosomal subunits, probably by chaperoning protein interactions during the export process.

Enhancement of hemoglobin and F-cell production by targeting growth inhibition and differentiation of K562 cells with ribonucleotide reductase inhibitors (didox and trimidox) in combination with streptozotocin.

Upon appropriate drug treatment, the human erythroleukemic K562 cells have been shown to produce hemoglobin and F‐cells. Fetal hemoglobin (Hb F) inhibits the polymerization events of sickle hemoglobin (Hb S), thereby ameliorating the clinical symptoms of sickle cell disease. Ribonucleotide reductase inhibitors (RRIs) have been shown to inhibit the growth of myeloid leukemia cells leading to the production of Hb F upon differentiation. Of the RRIs currently in use, hydroxyurea is the most effective agent for Hb F induction. We have examined the capacity of two novel RRIs, didox (DI) and trimidox (TRI), in combination with streptozotocin (STZ), to induce hemoglobin and F‐cell production. The K562 cells were cultured with different concentrations of didox‐STZ or trimidox‐STZ at a fixed molar ratio of 3:1 and 1:5 for 96 hr, respectively. At pre‐determined time intervals, aliquots of cells were obtained and total hemoglobin (benzidine positive) levels, number of F‐cells, and Hb F were determined by the differential staining technique, fetal hemoglobin assay kit, and fluorescence cytometry respectively. The effect of combined drug treatment on the growth of K562 cells was examined by isobologram analysis. Our results indicate that a synergistic growth‐inhibitory differentiation effect occurred when didox or trimidox was used in combination with STZ on K562 cells. There was an increase in the number of both benzidine‐positive normoblasts and F‐cells, accompanied by morphologic appearances typical of erythroid maturation. On day 4, the number of benzidine‐positive cells showed a 6–9‐fold increase and the number of F‐cells was between 2.5‐ and 5.7‐fold higher than the respective controls. Based upon these results, treatment with a ribonucleotide reductase inhibitor, such as didox or trimidox, in combination with STZ, might offer an additional promising option in sickle cell disease therapy. Am. J. Hematol. 63:176–183, 2000.

EVIDENCE THAT THE NOVOBIOCIN-SENSITIVE ATP-BINDING SITE OF THE HEAT SHOCK PROTEIN 90 (HSP90) IS NECESSARY FOR ITS AUTOPHOSPHORYLATION

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N‐terminal domain of Hsp90 has been shown to contain an unusual ATP‐binding site. A well‐known inhibitor of Hsp90 function is geldanamycin binding to the N‐terminal ATP‐binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP‐binding site in the C‐terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with γ32P‐ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C‐terminal ATP‐binding site is required for autophosphorylation of Hsp90.

Reagents suitable for the crosslinking of nucleic acids to proteins

Abstract The synthesis of 3-(4-bromo-3-oxobutane-1-sulphonyl)propionic acid p -nitrophenyl ester, of the corresponding N -hydroxysuccinimide ester, and of p -azidophenylacetic imidoester is described. The first two compounds may be attached to adenine and cytosine in nucleic acid-protein mixtures at pH 6.0 preferentially, although in competition with side reactions to amino acid side chains, and then to protein in a second step at pH 8. The third compound rapidly reacts with protein lysine amino groups at pH 7.8, and, after photoactivation of the azido group, shows a preference in the second step reaction for the aromatic bases of the nucleic acid in the mixture.

Bile acid binding proteins in hepatocellular membranes of newborn and adult rats. Identification of transport proteins with azidobenzamidotauro[14C]cholate ([14C]ABATC)

Neonatal hepatocytes are less active in uptake of bile acids than are mature hepatocytes. This phenomenon has been further investigated by transport studies with azidobenzamidotaurocholate (ABATC). Taurocholate, cholate and the photolabile ABATC were taken up by liver cells of adult rats by a sodium-dependent and by an additional sodium-independent mechanism. In the dark, ABATC inhibited the uptake of taurocholate and cholate. Taurocholate decreased the transport of ABATC in a competitive manner, both in the presence and absence of sodium. In neonatal hepatocytes the Vmax for taurocholate and for ABATC was similar but was lower than in mature liver cells. In contrast, the Km was similar for neonatal and mature hepatocytes. For identification of binding proteins in both kinds of cells ABATC was photolysed after preincubation with isolated hepatocytes. Under our experimental conditions (single ultraviolet flash) about 80% of the azido groups was converted to nitrene. The covalently binding nitrene derivative inhibited bile salt transport irreversibly. Photolabeling of intact hepatocytes or of isolated plasma membranes with ABATC resulted in radioindication of membrane proteins with 67, 60, 54, 50 and 43 kDa in mature plasma membranes but of proteins with masses of 67, 54, 43 and 37 kDa in neonatal basolateral membranes. The 50 kDa protein in largely lacking in membranes of 9-day-old rats. The process of photolabeling itself was sodium-independent when isolated cells were treated with ABATC. In contrast, the degree of labeling of intact hepatocytes was markedly reduced in the absence of sodium and chloride. 100-fold molar excess of taurocholate, benzamidotaurocholate (BATC), phalloidin or cyclosomatostatin protected isolated plasma membranes against coupling of ABATC. Photolabeling of hepatoma cells known to be deficient in bile salt transport did not result in radiomodification of membrane proteins.

Identification of the D-glucose binding polypeptide of the renal Na+-D-glucose cotransporter with a covalently binding D-glucose analog.

Affinity labeling D‐Glucose analog D‐Glucose binding site Na±D‐glucose cotransporter Brush‐border membrane Kidney