ATP-dependent Proteolytic Activity Research Articles

Oxidative post-translational modifications develop LONP1 dysfunction in pressure overload heart failure.

Mitochondrial compromise is a fundamental contributor to heart failure. Recent studies have revealed that several surveillance systems maintain mitochondrial integrity. The present study evaluated the role of mitochondrial AAA+ protease in a mouse model of pressure overload heart failure. The fluorescein isothiocyanate casein assay and immunoblotting for endogenous mitochondrial proteins revealed a marked reduction in ATP-dependent proteolytic activity in failing heart mitochondria. The level of reduced cysteine was decreased, and tyrosine nitration and protein carbonylation were promoted in Lon protease homolog (LONP1), the most abundant mitochondrial AAA+ protease, in heart failure. Comprehensive analysis revealed that electron transport chain protein levels were increased even with a reduction in the expression of their corresponding mRNAs in heart failure, which indicated decreased protein turnover and resulted in the accumulation of oxidative damage in the electron transport chain. The induction of mitochondria-targeted human catalase ameliorated proteolytic activity and protein homeostasis in the electron transport chain, leading to improvements in mitochondrial energetics and cardiac contractility even during the late stage of pressure overload. Moreover, the infusion of mitoTEMPO, a mitochondria-targeted superoxide dismutase mimetic, recovered oxidative modifications of LONP1 and improved mitochondrial respiration capacity and cardiac function. The in vivo small interfering RNA repression of LONP1 partially canceled the protective effects of mitochondria-targeted human catalase induction and mitoTEMPO infusion. Oxidative post-translational modifications attenuate mitochondrial AAA+ protease activity, which is involved in impaired electron transport chain protein homeostasis, mitochondrial respiration deficiency, and left ventricular contractile dysfunction. Oxidatively inactivated proteases may be an endogenous target for mitoTEMPO treatment in pressure overload heart failure.

Loss of the m-AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation

The m-AAA protease subunit AFG₃L₂ is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG₃L₂ are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG₃L₂ causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG₃L₂ is still unclear. Here, we show that depletion of AFG₃L₂ leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG₃L₂ in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG₃L₂-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG₃L₂.

Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion

Prooxidents can induce reversible inhibition or irreversible inactivation and degradation of the mitochondrial enzyme aconitase. Cardiac ischemia/reperfusion is associated with an increase in mitochondrial free radical production. In the current study, the effects of reperfusion-induced production of prooxidants on mitochondrial aconitase and proteolytic activity were determined to assess whether alterations represented a regulated response to changes in redox status or oxidative damage. Evidence is provided that ATP-dependent proteolytic activity increased during early reperfusion followed by a time-dependent reduction in activity to control levels. These alterations in proteolytic activity paralleled an increase and subsequent decrease in the level of oxidatively modified protein. In vitro data supports a role for prooxidants in the activation of ATP-dependent proteolytic activity. Despite inhibition during early periods of reperfusion, aconitase was not degraded under the conditions of these experiments. Aconitase activity exhibited a decline in activity followed by reactivation during cardiac reperfusion. Loss and regain in activity involved reversible sulfhydryl modification. Aconitase was found to associate with the iron binding protein frataxin exclusively during reperfusion. In vitro, frataxin has been shown to protect aconitase from [4Fe-4S](2+) cluster disassembly, irreversible inactivation, and, potentially, degradation. Thus, the response of mitochondrial aconitase and ATP-dependent proteolytic activity to reperfusion-induced prooxidant production appears to be a regulated event that would be expected to reduce irreparable damage to the mitochondria.

Functional Proteolytic Complexes of the Human Mitochondrial ATP-dependent Protease, hClpXP

Human mitochondrial ClpP (hClpP) and ClpX (hClpX) were separately cloned, and the expressed proteins were purified. Electron microscopy confirmed that hClpP forms heptameric rings and that hClpX forms a hexameric ring. Complexes of a double heptameric ring of hClpP with hexameric hClpX rings bound on each side are stable in the presence of ATP or adenosine 5'-(3-thiotriphosphate) (ATPgammaS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPgammaS-dependent peptidase activity. hClpXP cannot degrade lambdaO protein or GFP-SsrA, specific protein substrates recognized by Escherichia coli (e) ClpXP. However, eClpX interacts with hClpP, and, when examined by electron microscopy, the resulting heterologous complexes are indistinguishable from homologous eClpXP complexes. The hybrid eClpX-hClpP complexes degrade eClpX-specific protein substrates. In contrast, eClpA can neither associate with nor activate hClpP. hClpP has an extra C-terminal extension of 28 amino acids. A mutant lacking this C-terminal extension interacts more tightly with both hClpX and eClpX and shows enhanced enzymatic activities but still does not interact with eClpA. Our results establish that human ClpX and ClpP constitute a bone fide ATP-dependent protease and confirm that substrate selection, which differs between human and E. coli ClpX, is dependent solely on the Clp ATPase. Our data also indicate that human ClpP has conserved sites required for interaction with eClpX but not eClpA, implying that the modes of interaction with ClpP may not be identical for ClpA and ClpX.

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease.

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.

The Brucella abortus Lon functions as a generalized stress response protease and is required for wild-type virulence in BALB/c mice.

The gene encoding a Lon protease homologue has been cloned from Brucella abortus. The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB:lacZ fusion to wild-type levels. A sigma32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses. ATP-dependent proteolytic activity was also demonstrated for purified recombinant Lon. To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange. In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41 degrees C and displayed an increased sensitivity to killing by puromycin and H2O2. GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain. Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice. These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild-type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.

Induction of acclimative proteolysis of the light-harvesting chlorophyll a/b protein of photosystem II in response to elevated light intensities

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Effects of the Cys Mutations on Structure and Function of the ATP-dependent HslVU Protease in Escherichia coli: THE CYS287 TO VAL MUTATION IN HslU UNCOUPLES THE ATP-DEPENDENT PROTEOLYSIS BY HslVU FROM ATP HYDROLYSIS

To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.

Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues

Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca 2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40 000×g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4–80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3–4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15–20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin- 125I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.

Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY

ClpQ (HsIV) is a homolog of the β-subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HsIU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit M r 19 000) and ClpY (subunit M r 49 000) were purified separately as oligomeric proteins with molecular weights of ∼220 000 and ∼350 000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP-dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six-fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry-matched ClpYQ system.

ATP-dependent proteolysis in yeast mitochondria.

An ATP-dependent proteolysis system in yeast mitochondria was characterized by examining the hydrolysis of mitochondrial translation products in isolated mitochondria. Degradation of [35S]methionine-labeled polypeptides synthesized in isolated yeast mitochondria was activated by exogenously added ATP. ADP, GTP, and CTP substituted for ATP to some extent, but nonhydrolyzable ATP analogues did not. Adenosine-5'-O-(3'-thio-triphosphate) effectively competed with ATP as activator. Carboxyatractyloside, an inhibitor of adenine nucleotide translocation across the mitochondrial inner membrane, and the metal chelator o-phenanthroline inhibited the ATP-dependent proteolysis. The latter inhibition was abolished by subsequent addition of Mn2+ or Co2+ but not Ca2+ or Zn2+. Hemin inhibited the ATP-dependent proteolysis of mitochondrial translation products with a half-maximum inhibition at 12 microM. Analysis by SDS-polyacrylamide gel electrophoresis showed that [35S]methionine-labeled polypeptides were rapidly degraded into low-molecular-weight species. Submitochondrial particles retained the ATP-dependent proteolytic activity and had the same spectrum of inhibitors as intact mitochondria except for a reduced effect of carboxyatractyloside. These results indicate that yeast mitochondria contain an ATP-dependent and hemin-sensitive proteolysis system which is associated with the inner membrane and can hydrolyze mitochondrial translation products, and that a chelator-sensitive protease is probably involved in this system.

Requirement for the Yeast Gene LON in Intramitochondrial Proteolysis and Maintenance of Respiration

The role of protein degradation in mitochondrial homeostasis was explored by cloning of a gene from Saccharomyces cerevisiae that encodes a protein resembling the adenosine triphosphate (ATP)-dependent bacterial protease Lon. The predicted yeast protein has a typical mitochondrial matrix-targeting sequence at its amino terminus. Yeast cells lacking a functional LON gene contained a nonfunctional mitochondrial genome, were respiratory-deficient, and lacked an ATP-dependent proteolytic activity present in the mitochondria of Lon+ cells. Lon- cells were also impaired in their ability to catalyze the energy-dependent degradation of several mitochondrial matrix proteins and they accumulated electron-dense inclusions in their mitochondrial matrix.

Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease

Lon protease from Escherichia coli is an ATP-dependent protease which plays important roles in regulating the levels of specific proteins and in eliminating abnormal proteins. A major problem of working with Lon protease, the inability to substantially overproduce the enzyme, has been overcome by placing the lon gene under the control of an inducible trp promoter within a copy-number-controllable plasmid. Induction resulted in higher levels of production of the protease ( ~ 100 μg/ml of cell culture) than were previously possible. The enzyme has been purified to apparent homogeneity and shown to possess the characteristic ATP-dependent proteolytic activity. Sequence verification during DNA manipulations revealed differences from two previously published sequences for the lon gene.

An analysis of the effect of changes in growth temperature on proteolysis in vivo in the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven protease separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 degrees C after a shift-up from 0 degrees C increased to a level 36% and 17% higher than that of cells grown at 0 degrees C and 13 degrees C, respectively. A shift-down to 0 degrees C from 13 degrees C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 degrees C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 degrees C, but a decrease at 35 degrees C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.

Proteolytic response to the expression of an abnormal beta-galactosidase in Escherichia coli.

Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant beta-galactosidase was investigated. A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome. Induction of expression of the mutant beta-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity. Growth at temperatures above 40 degrees C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37 degrees C, and the ATP-dependent activity increased 2.5-fold from 30 to 42 degrees C. Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal beta-galactosidase at 37 degrees C, but no such response was evident when mutant beta-galactosidase expression was induced at 30 degrees C. In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid. Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity. These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E. coli. These responses were reduced by induction at lower temperatures.

A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex).

Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.

Rat retina has an active and stable ubiquitin-protein conjugating system.

We describe here the presence of ubiquitin and its conjugation system in the rat retina. Retinal homogenates and supernatants conjugate [125I]human ubiquitin with either endogenous or exogenous proteins. The conjugating activity is relatively stable over time, requires ATP, and has a pH optimum of approximately 8. The most prominent [125I]ubiquitin conjugates formed are larger than 130 kDa. Several other minor conjugates are also formed between the molecular weights 17 and 75 kDa. The endogenous levels of free and conjugated forms of ubiquitin have been determined in the rat retina. More than 50% of retinal ubiquitin is covalently bound to target proteins. In addition, activities responsible for the ATP-dependent degradation and disassembly of both endogenous and exogenous ubiquitin conjugates have been detected in vitro. These results provide evidence that the retina contains active and stable ubiquitin-conjugating enzymes that recognize retinal proteins and have ATP-dependent proteolytic activity.

Increased ATP-dependent proteolytic activity in lon-deficient Escherichia coli strains lacking the DnaK protein

Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated protease. Comparison of the levels of ATP-dependent proteolytic activity present in lon rpoH dnaK mutants and in lon rpoH dnaK+ mutants showed that the level of ATP-dependent proteolytic activity was elevated in the lon rpoH dnaK mutant strain. These findings suggest that DnaK negatively regulates a new ATP-dependent proteolytic activity, independently of sigma 32. Other results indicate that an ATP-dependent proteolytic activity was increased in a lon alp strain after heat shock. It is not yet known whether the same protease is associated with the increased ATP-dependent proteolytic activity in the dnaK mutants and in the heat-shocked lon alph strain.

Identification of a ubiquitin- and ATP-dependent protein degradation pathway in rat cerebral cortex

To investigate the existence of a ubiquitin-dependent protein degradation system in the brain, the proteolytic activity of the cerebral cortex was examined. The soluble extract of rat cerebral cortex degraded 125I-radiolabeled lysozyme in an ATP-dependent manner. The ATP-dependent proteolysis was suppressed with iodoacetamide, which inhibits ubiquitin conjugation, and was abolished by blocking of the amino residues of lysozyme. These results suggest the participation of ubiquitination in the proteolytic activity. An ATP-dependent 125I-ubiquitin-conjugating activity was detected in fraction II from the cerebral cortex. The presence of ATP-dependent proteolytic activity which acted preferentially on ubiquitinated lysozyme was demonstrated, using ubiquitin-125I-lysozyme conjugates as a substrate. The proteinase had a molecular mass of 1500 kDa and displayed nucleotide dependence and sensitivity to various proteinase inhibitors similar to those of the 26S proteinase complex found in reticulocytes. Dialysis of the soluble fraction caused a decrease in the proteolytic activity of ATP-dependent and preferential for ubiquitin-lysozyme conjugates and a reciprocal increase in the ATP-independent free 125I-lysozyme-degrading activity which was scarcely detected before dialysis. The former ATP-dependent proteolytic activity may play a physiological role in the brain.

Induction of the ATP-dependent proteolytic system in guinea pig reticulocyte lysates by triiodothyronine

The mechanism involved in the decreased numbers of several trans-membrane proteins such as sodium pump sites, sodium-lithium countertransport, sodium potassium cotransport proteins, proteins mediating the passive efflux of sodium and insulin receptors in erythrocytes from patients with hyperthyroidism is not known. The ATP-dependent proteolytic system which is involved in the loss of trans-membrane proteins during the maturation of the reticulocyte may be involved in the accelerated loss of these membrane proteins. Therefore, the effect of thyroid hormones on the ATP-dependent proteolytic activity of reticulocyte lysates was examined in this study. Reticulocytosis was induced in 14 guinea pigs by phenylhydrazine hydrochloride injections for 5 consecutive days followed by 2 days of rest. T 3 (10μg/100g body weight) was injected into 7 animals on day 4 and day 6. Reticulocyte-rich blood was withdrawn on day 8. Oxygen consumption determined 24 hours after injection of T 3 was 25% higher (p<0.01) and T 3 treated animals had a 2.5 fold higher (p<0.01) weight loss than control animals. The ATP-dependent proteolytic activity measured in reticulocyte lysates using 125I labelled lysozyme was 3.6 fold higher in the T 3 than in the control group of guinea pigs (p<0.01). We conclude that thyroid hormones induce the ATP-dependent proteolytic activity of reticulocyte lysates which may be responsible for the reduced number of several trans-membrane proteins found in erythrocytes from patients with hyperthyroidism.