Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.     

Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis

To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin-inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme.     

A particle-associated ATP-dependent proteolytic activity in erythroleukemia cells

An ATP-dependent proteolytic activity has been detected in both mouse erythroleukemia (Friend) cells and human (K562) erythroleukemia cells. Exposure of the Friend cells to dimethyl sulfoxide, which stimulates differentiation, increased ATP-dependent proteolysis approximately 2-fold although inducing differentiation in the K562 line had no significant effect on proteolysis. In contrast to the previously described soluble ATP-dependent proteolytic system of reticulocytes, the activity in the more primative erythroid cells is associated with a particulate fraction and is readily sedimentable by centrifugation at 100,000 X g for 1 h. Like the soluble reticulocyte system, the particulate activity requires divalent cation and is inhibited by hemin as well as vanadate. The activity was isolated on a sucrose cushion (30%) and did not appear to be associated with membranes, cytoskeleton, or polysomes. This enzymatic activity which degrades abnormal globin chains may initially reside in a particulate fraction and then become solubilized during erythroid maturation to the reticulocyte stage. Alternatively, the particulate activity may disappear with cell maturation being replaced by a distinct soluble activity. ATP-dependent proteolytic activity is eventually lost with reticulocyte maturation and further aging of erythrocytes.     

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 Ca²⁺ 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.     

Glial Fibrillary Acidic Protein in the Cytoskeletal and Soluble Protein Fractions of the Developing Rat Brain

The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme-linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel ATP-requiring protease from skeletal muscle that hydrolyzes non-ubiquitinated proteins

Previously, we isolated an ATP-dependent proteolytic pathway in muscle, liver, and reticulocytes that requires ubiquitin and the enzymes which conjugate ubiquitin to proteins. We report here that skeletal muscle contains another soluble alkaline energy-dependent (but ubiquitin-independent) proteolytic activity. The cleavage of non-ubiquitinated protein substrates by the partially purified protease requires ATP hydrolysis since ATP in the absence of Mg2+, nonhydrolyzable ATP analogs, and pyrophosphate all fail to stimulate proteolysis. Proteolytic activity is also stimulated by UTP, CTP, and GTP, although not as effectively as by ATP (Km(ATP) = 0.027 mM). The enzyme is inactivated by the serine protease inhibitors diisopropyl fluorophosphate and 3,4-dichloroisocoumarin, but not by specific inhibitors of aspartic, thiol, or metalloproteases. It is maximally active at pH 8 and has a molecular weight of approximately 600,000. This new activity differs from the 720-kDa multicatalytic proteinase, but resembles the soluble ATP-dependent proteolytic system that we previously isolated from murine erythroleukemia cells.     

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. T3 (10 micrograms/100 g 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 T3 was 25% higher (p less than 0.01) and T3 treated animals had a 2.5 fold higher (p less than 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 T3 than in the control group of guinea pigs (p less than 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.     

A cysteine metalloproteinase from mouse liver cytosol

A cysteine metalloproteinase that degrades 125I-insulin B chain at neutral pH values was isolated from C3H mouse liver. The enzyme was partially purified from the 100,000g supernatant fraction by ammonium sulfate precipitation, DEAE-cellulose chromatography, and fast protein liquid chromatography. The molecular weight of the proteinase was estimated to be 190,000 by gel filtration on Sephadex G-200. Degradation of 125I-insulin B chain by the proteinase was inhibited by p-hydroxymercuribenzoate (PHMB) and iodoacetate (cysteine proteinase inhibitors) and by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (metalloproteinase inhibitors). The proteinase also degraded 125I-glucagon but did not hydrolyze 125I-insulin, leucine-2-naphthylamide, or several large proteins. Equivalent levels of EDTA- and PHMB-inhibitable 125I-insulin B chain-degrading activity were observed in the 100,000g supernatant fractions of brain, liver, lung, kidney, heart, and spleen from four mouse strains (C3H/HeN, CBA/J, ICR, and C57BL/6). High levels of 125I-insulin B chain-degrading activity were found in the particulate fraction of kidneys and lungs from these four mouse strains; these activities were inhibited by EDTA but not by PHMB. The activity of the soluble liver cysteine metalloproteinase was not altered in C3H mice treated ip with metal chelators, bacterial endotoxin, phenobarbital, dexamethasone, or insulin. Starvation for 24 or 48 hr and alloxan-induced diabetes diminished total activity of this enzyme in liver by about 50 and 30%, respectively. This soluble polypeptide-degrading enzyme appears to be ubiquitous in mice and to be regulated by nutritional conditions.     

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.     

The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin-conjugated proteins

Mammalian cells contain two large proteolytic complexes, the 650-kDa proteasome (or multicatalytic protease) and the 1500-kDa (26 S) Ubiquitin-conjugate-degrading enzyme. Since the proteasome is also required for the ATP-dependent degradation of ubiquitinated proteins, we tested whether it may be a component of the larger complex. The proteasome normally is soluble in 38% ammonium sulfate. However, after preincubation of reticulocyte extracts with ATP, several proteasome activities appeared in the 38% ammonium sulfate pellet, including the ability to degrade hydrophobic peptides and 14C-casein. Also, following preincubation with ATP, the precipitable fraction could degrade 125I-lysozyme-ubiquitin (Ub) conjugates. The activities were not present after incubation without ATP or with a nonmetabolizable ATP analog. Nondenaturing gel electrophoresis indicated the ATP-dependent appearance of a new band which degraded proteasome substrates, and reacted with an anti-proteasome monoclonal antibody on Western blot. This new band appeared larger than the proteasome and migrated similarly to the larger Ub-conjugate-degrading complex. The formation of the larger complex required factor(s) present in the 38% ammonium sulfate pellet and either the 40-80% fraction or the purified proteasome from reticulocytes or muscle. After complex formation, hydrolysis of Ub-protein conjugates and also the non-ubiquitinated substrate, casein, was stimulated severalfold by ATP, but non-metabolizable ATP analogs had little or no effect. Thus, the proteasome corresponds to component CF-3 of Ganoth et al. (Ganoth, D., Leshinisky, E., Eytan, E., and Hershkov, A. (1989) J. Biol. Chem. 263 12412-12419) and undergoes an energy-dependent association with other factors to form the 1500-kDa, ATP-requiring proteolytic complex.     

Polyamines inhibit the ATP-dependent proteolytic pathway in rabbit reticulocyte lysates

Reticulocytes contain a soluble nonlysosomal proteolytic pathway that requires ATP and ubiquitin. Polyamines at physiological concentrations were found to inhibit rapidly the ATP-dependent proteolytic system in reticulocyte lysates; spermidine and putrescine inhibited this process by 26-72% and spermine by 71-96%. Spermine had little effect on the ATP-independent breakdown of oxidant-treated hemoglobin. By fractionating the ATP-dependent system, we show that polyamines inhibit the ATP-dependent degradation of ubiquitin-protein conjugates.     

The eye lens has an active ubiquitin-protein conjugation system

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.     

A comparative study of soluble calcium-dependent proteolytic activity in brain

Recent studies have shown that soluble calcium activated proteases (calpains) in brain degrade proteins associated with the cytoskeleton and vary markedly in activity across regions and as a function of development. It was suggested that the observed differences in calpain activity reflect differences in the turnover rate of structural elements. The present study extends this analysis by measuring the properties and activity of calpain in representatives of the five classes of vertebrates with particular emphasis on the mammals. No evidence for proteolysis was found in soluble fractions of fish brains at neutral pH in the presence or absence of added calcium. A substantial calcium-independent proteolytic activity was found in amphibian brains—the effects of a variety of protease inhibitors indicated that it is also a neurtral thiol (cysteine) protease. Reptilian brains exhibited both calcium-independent and calcium-dependent proteolytic activity. Virtually all proteolytic activity in birds (5 species) and mammals (9 species) measured at neutral pH was calcium-dependent. The endogenous substrates for the calcium activated proteases were very similar in several species of birds and mammals as were the effects of a variety of protease inhibitors. However, the activity of the enzyme, expressed per mg of soluble protein, was highly and negatively correlated with brain size in the mammals. The allometric expression for this relationship was similar to that found for the density of neurons in cerebral cortex as a function of absolute brain size. These results indicate that soluble proteolytic enzymes in brain are differentially expressed among classes of vertebrates and suggest that the turnover of cytoskeletal elements in birds and mammals differs in important ways from that found in fish and amphibians. The results obtained for mammals raise the possibility of a relationship between brain size and the rate at which structural elements are broken down and replaced in this vertebrate class.     

Genotype-dependent proteolytic response of spring wheat to water deficiency.

Changes in proteolytic activities in response to water deficiency have been investigated in ten genotypes of spring wheat (Triticum aestivum L.) differing in response to water deficit stress and ability to acclimate. To determine subcellular localization and the type of proteases, mesophyll protoplasts isolated from wheat leaves were purified. Proteolytic activities were assayed using azocasein in the case of vacuolar proteinases at pH 5.0 and 125I-lysozyme in the case of extravacuolar ATP-dependent proteinases at pH 8.2. ATP-dependent proteolytic activity was found to be confined to the extravacuolar fraction while the azocaseinolytic activity to vacuoles. Dehydration increased vacuolar azocaseinolytic activity at both stages of plant development (shooting and heading), but the increase was significantly lower in more tolerant genotypes. The extravacuolar energy-dependent 125I-lysozyme degradation was low at the shooting stage but it was higher in the genotypes with a greater critical water saturation deficit. At the heading phase in the non-acclimated flag leaves ATP-dependent 125I-lysozyme degradation decreased in a genotype-dependent manner, but was enhanced upon acclimation to the same extent irrespective to the genotype ability to acquire dehydration tolerance during acclimation. The results presented indicate that both pathways of protein degradation are interlinked upon dehydration and are genotype dependent.     

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex

1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.     

Ubiquitin conjugation to endogenous proteins in the dormant tuber of Helianthus tuberosus and during the first cell cycle

Ubiquitin is a small protein involved in an ATP-dependent proteolytic pathway in all eukaryotes. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. We have investigated the presence of ubiquitin (Ub) and the ubiquitin-conjugating system in dormant and activated tubers of Helianthus tuberosus L. cv. OB 1 that represent a widely used model system for studies on the cell cycle in plants. Immunoblot experiments revealed the presence of free ubiquitin and ubiquitin conjugates. Furthermore, the presence of an active ubiquitin-conjugating system, both time- and ATP-dependent, was demonstrated by incubation with ¹²⁵I-labeled ubiquitin. A few proteins able to form thiol esters with ¹²⁵I-Ub and probably corresponding to ubiquitin-conjugating enzymes, E1 and E2s, were also found. During the first cell cycle, several proteins become ubiquitinated. In particular a large amount of protein conjugates was present at 6 h when the lowest content of free ubiquitin was found. Subsequently, a dramatic decrease in ubiquitin conjugates occurred. It is well known that cell cycle progression in eukaryotes depends on cyclin levels and cyclin B degradation is ubiquitin- and ATP-dependent. By immunoblot experiments we showed that cyclin B in H. tuberosus is present as at least two protein bands of 50 and 54 kDa and that their amounts undergo profound changes during the cell cycle. The 54-kDa band was also recognized by an anti-ubiquitin antibody. These data seem to indicate that in H. tuberosus activated tuber slices, the ATP-dependent ubiquitin proteolytic pathway is involved in the dedifferentiation process occurring after the artificial break of dormancy when the cells acquire the characteristics linked to the meristematic state.