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A Hershko

Publications and source records attributed to A Hershko.

At least 73 records · Page 4Linked to original sources
PubMed1982

"Covalent affinity" purification of ubiquitin-activating enzyme.

We have previously described an enzyme that activates ubiquitin, the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes (Ciechanover, A., Heller, H., Katz-Etzion, R., and Hershko, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 761-765). It carries out ubiquitin-dependent PPi-ATP and AMP-ATP exchange reactions and binds to the activated polypeptide by a thiolester linkage. We describe here a procedure for the purification of this enzyme by its binding to ubiquitin-Sepharose. Binding of the enzyme to the affinity column requires ATP and Mg2+, and bound enzyme cannot be displaced by high salt but can be eluted by raising the pH, by increased concentrations of a thiol compound, or by the joint supplementation of AMP and pyrophosphate. Another form of the enzyme that cannot carry out AMP-ATP exchange (but catalyzes ubiquitin-dependent PPi-ATP exchange) does not bind to the affinity column. These data indicate that a covalent, possibly thiolester intermediate, is formed between the activating enzyme and Sepharose-bound ubiquitin. It is suggested designating this procedure of enzyme isolation "covalent affinity" chromatography. The purified enzyme has an apparent Mr = 210,000 and appears to be composed of two subunits of Mr = 105, 000. ATP-dependent binding of ubiquitin to the purified enzyme and to its subunit is demonstrated.

Animals↗
PubMed1982

Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation.

Energy-dependent proteolysis in rabbit reticulocyte lysates has an absolute requirement for both ATP and ubiquitin that is believed due to the ATP-dependent covalent conjugation of ubiquitin (Ub) to substrate proteins as a signal event. Recently the enzyme responsible for activation of ubiquitin has been purified to near homogeneity by covalent affinity chromatography. In the present work we demonstrate that the enzyme catalyzes the net reaction (formula: see text); in which both an enzyme-bound COOH-terminal ubiquitin thiolester and a COOH-terminal ubiquitin adenylate are formed. The complex is sufficiently stable to be isolated by exclusion chromatography and contains ubiquitin/AMP in a ratio of 2. The presence of two forms of covalently linked ubiquitin provides a rationale for the observation of both ubiquitin-dependent ATP:PPi and ATP:AMP exchange. Selective derivatization of the thiol site with iodoacetamide results in a modified enzyme capable of catalyzing only ATP:PPi exchange and in forming only ubiquitin adenylate. Derivatization of the thiol site prevents conjugate formation in a reconstituted system, demonstrating that the ubiquitin thiolester is the proximal donor for subsequent conjugate formation. A three-step mechanism is proposed for ubiquitin activation by the enzyme prior to conjugation.

Animals↗
PubMed1981

Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown.

The heat-stable polypeptide of the ATP-dependent proteolytic system was previously found to form covalent conjugates with proteins and to be activated by ATP in an adenylylation mechanism. To identify the functional amino acid of the polypeptide, the activated residue was specifically labeled by the reductive cleavage of the intermediate with [3H]borohydride. Following acid hydrolysis, the reduced labeled derivative was found to be completely oxidizable by periodate with formation of [3H]formaldehyde, and was identified as ethanolamine by thin layer chromatography, electrophoresis, and amino acid analyzer chromatography. These results indicate that the activated amino acid residue of the polypeptide is COOH-terminal glycine.

Adenosine Triphosphate↗
PubMed1981

Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester.

Adenosine Triphosphate↗
PubMed1980

Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes.

A heat-stable polypeptide from rabbit reticulocytes has been previously shown to be required for ATP-dependent protein breakdown (Ciechanover, A., Hod, Y., and Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105) and to form covalent conjugates with proteins in an ATP-requiring reaction (Ciechanover, A., Heller, H., Elias, S., Haas, A.L., and Hershko, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1365-1368). We now describe its purification, characterization, and tissue distribution. Its presence in erythrocytes at high level makes this a possible preferred source in the future.

Amino Acids↗
PubMed1980

ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation.

The heat-stable polypeptide (APF-1) required for ATP-dependent proteolysis in reticulocytes enters into high molecular weight conjugates upon incubation with the fraction of reticulocytes that is retained by DEAE-cellulose. Conjugate formation requires ATP and Mg2+ and its inhibited by N-ethylmaleimide. UTP and GTP are inactive. These properties are identical to those of ATP-dependent protein breakdown in the same system, suggesting that the conjugates are intermediates in this process. The APF-1 conjugates are stable in sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Sephadex G-75 isolation and are resistant to mild acid, alkali, heat denaturation, and reduction; the conjugates are therefore covalent.

Adenosine Triphosphate↗
PubMed1980

Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis. Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or alpha-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP.

Adenosine Triphosphate↗
PubMed1979

A high molecular weight protease in liver cytosol.

A high molecular weight (greater than 400,000) protease active with [3H]leucine-labeled globin has been found in the postmicrosomal fraction of mouse kidney, brain, heart, spleen, and tumor cells and is most active in liver. The presence in liver was unexpected because liver cytosol is very ineffective in the breakdown of endogenous, labeled proteins. The enzyme has a number of properties that distinguish it from known cathepsins in addition to its high molecular weight. It is most active at pH approximately 7.5. When purified, it is unstable above 20 degrees C and is stabilized by metal chelating agents such as citrate, creatine-P, and glycerate-3-P. It is an -SH protease, but its thermal instability is not affected by 1 mM dithiothreitol. The enzyme is not lysosomal.

Animals↗
PubMed1979

Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP.

The ATP-dependent proteolytic cell-free system from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of 125I-labeled bovine serum albumin radioactivity. In addition to the previously reported heat-stable polypeptide [Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105], we now describe a protein of high molecular weight (approximately 450,000) that is labile at 42 degrees C. The extremely heat-labile factors is remarkably stabilized by ATP. GTP and CTP, which do not stimulate protolysis, do not stabilize this factor. Adenylate nucleotides such as ADP or the nonhydrolyzable beta,gamma imido or methylene analogues of ATP cause stabilization although they do not activate proteolysis. A third protein component of the protease system, stable at 42 degrees C, has been separated from the heat-labile species by salt precipitation. All three components are required with ATP for proteolytic activity, but thus far only the heat-labile factor has been shown to interact directly with ATP.

Adenosine Triphosphate↗
PubMed1978

Protein turnover in senescent cultured chick embryo fibroblasts.

The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.

Cell Count↗
PubMed1977

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells.

Corticosteroid-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 X g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I leads to II leads to III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cell subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.

Carcinoma, Hepatocellular↗
PubMed1976

Relationship of the pool of intracellular valine to protein synthesis and degradation in cultured cells.

To explore the role of the pool of intracellular free valine in the processes of protein synthesis and protein degradation, cultured hepatoma (HTC) cells were incubated in media containing varying concentrations of L-valine, under conditions of constant rates of protein synthesis and protein breakdown, and at steady state levels of intracellular valine specific radioactivities. Two types of experiments were compared: in the first (designated "incorporation experiment"), unlabeled cells were exposed to [3H]valine for a short period of time. In the second (termed "reincorporation experiment"), cells were prelabled with [3H]valine and then incubated for a brief period with media containing different concentrations of unlabeled valine; reincorporation of [3H]valine was calculated by the difference between the release of [3H]valine from labeled cellular proteins at low valine concentrations, and the maximal rate of the release at high valine concentrations. In both types of experiments, the rates of [3H]valine incorporation or reincorporation were compared with the respective specific radioactivities of free intracellular valine. In the incorporation experiment, the rates of [3H]valine incorporation into protein calculated by the intracellular specific radioactivities were not constant, but showed an upward deviation at low valine concentrations. This is in agreement with the results of Mortimore, G.E., Woodside, K.H., and Henry, J.E. ((1972) J. Biol. Chem. 247, 2776-2784) in the perfused rat liver. By contrast, in the reincorporation experiment, the calculated rates of [3H]valine reincorporation based on intracellular specific radioactivities were constant throughout the range of valine concentrations. The constant value of calculated valine reincorporation was lower by 30 to 50% than the calculated rate of valine incorporation at high valine concentrations. The following model is proposed to explain these results. There is one common pool of free intracellular valine, but there are two sites where valyl-tRNA can be formed. The first is an internal site that utilizes valine from the intracellular pool, and the second is an external (possibly membranous) system that converts extracellular valine directly to valyl-tRNA. Valine originating from protein degradation flows into the intracellular pool, from which it can be reutilized by the internal system. According to these assumptions, in the incorporation experiment and at low valine concentrations, the specific activity of valyl-tRNA is higher than that of the intracellular pool of free valine, due to the contribution of the external system. On the other hand, in the reincorporation experiment the specific activity of extracellular valine is negligible in comparison with that of the intracellular pool. Therefore, in this case the specific activity of valyl-tRNA is proportional to that of the intracellular pool, with a constant dilution by unlabeled valine of extracellular origin...

Cell Line↗
PubMed1975

Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.

Carcinoma, Hepatocellular↗