Catalytic properties of three L-lactate dehydrogenases from saffron corms (Crocus sativus L)

Three L-lactate dehydrogenase isoenzymes were detected in saffron corms, using potassium ferricyanide as the electron acceptor. Their pH optima were 5.5, 7.5 and 9.5, respectively. All three dehydrogenases were substrate-inhibited by ferricyanide, but at different concentrations; maximum enzymatic activity was observed for 250, 100 and 600 M ferricyanide, at pH 5.5, 7.5 and 9.5, respectively. Catalytic efficiency, calculated per mg corm extract protein, was 1.9, 1.0 and 0.4 min^-1, respectively at pH 5.5, 7.5 and 9.5. Pseudo first order rate constant was also different under the three pH conditions. Malate was an inhibitor for the isoenzyme active at pH 9.5, but had no effect on the others.     

Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.

S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^?10M, Rh. glutinis acid protease; 1.8×10^?7M, Cladosporium acid protease; 3.0×10^?6M.

These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.

Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form.     

Phosphorylation of the carboxyl terminal region of dystrophin by mitogen-activated protein (MAP) kinase

Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy gene (DMD). The function of this protein remains to be elucidated. We have recently reported that dystrophin is phosphorylated,in vivo, in rat skeletal muscle primary cell culture (RE Milner, JL Busaan, CFB Holmes, JH Wang, M Michalak (1993) J Biol Chem 268: 21901–21905). This observation suggests that protein phosphorylation may have some role in modulating the function of dystrophin or its interaction with membrane associate dystroglycan. We report here that the carboxyl-terminal of dystrophin is phosphorylated by the MAP kinase p44^mpk (mitogen-activated protein kinase), from the sea star oocytes and by soluble extracts of rabbit skeletal muscle. Importantly we showed that native dystrophin in isolated sarcolemmal vesicles is phosphorylated by sea star p44^mpk. Partial purification and immunological analysis show that a mammalian kinase related to p44^mpk is present in the skeletal muscle extracts and that it contributes to phosphorylation of the carboxyl-terminal of dystrophin. This kinase phosphorylates dystrophin on a threonine residue(s). We conclude that phosphorylation of dystrophin may play an important role in the function of this cytoskeletal protein.Abbreviations MAP kinase mitogen-activated protein kinase - DMD Duchenne muscular dystrophy - GST Glutathione S-transferase - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MOPS 4-morpholinepropanesulfonic acid     

Properties of a calcium-activated protease in squid axoplasm which selectively degrades neurofilament proteins

Axoplasm extruded from the giant axon of the squid contains Ca²⁺-activated proteases. The protease in the 100,000X g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF₂₀₀), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca²⁺ ion, and no other divalent cation will substitute. The protease requires Ca²⁺ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF₂₀₀ appears to proceed through a 100,000 MW and possibly a 47,000–50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu²⁺ and Fe²⁺, sulphydryl inhibitors, and leupeptin. This specific Ca²⁺-activated protease in squid axoplasm has identical properties to Ca²⁺-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca²⁺-activated protease purified from human platelets effectively degrades squid NF₂₀₀, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1

The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (=0.058 h^–1) than at pH 6.5 (=0.028 h^–1). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.     

Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase

Purified human glucocerebrosidase isolated from placenta was modified with [¹⁴C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [¹⁴C]-carboxymethylated-cysteine identified as CM-Cys₁₈. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys₁₈. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with-mercaptoethanol followed by S-carboxymethylation with [¹⁴C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys₄-Cys₁₆, Cys₂₃-Cys₃₄₂, and Cys₁₂₆-Cys₂₄₈. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.     

Changes in composition and proteolytic enzyme activities of artemia during early development

• 1.1. The proximate composition, total and free amino acids, and proteases of Artemia nauplii were determined during early development.
• 2.2. Moisture increased from 71.0% to 80.8%, crude protein decreased from 13.2% to 8.8%, crude fat and ash varied slightly.
• 3.3. The total amino acids decreased. Free amino acids changed in three patterns.
• 4.4. Trypsin, chymotrypsin, carboxypeptidase A, B and cathepsin B and C increased in activity. The activity of trypsin was lower, while cathepsin B and C were the highest.
• 5.5. The protease activities were maximal at pH 7.5 and 8.0, and at 45°C on casein.
• 6.6. The optimal pH for carboxypeptidase A was 4.0, for carboxypeptidase B was 4.5, for trypsin and chymotrypsin were 7.0–7.5. The protease(s) active at pH 9.0–9.5 were to be determined.

     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

The effect of the dw gene on the muscle protein turnover rate in chickens

Fractional rates (%/day) of muscle protein synthesis and degradation of the genotypes Dw/Dw and dw/dw of male White Plymouth Rock chickens were determined by measuring the output of N-methylhistidine (N-MH) in the excreta at 2, 4, and 8 weeks of age. The fractional growth rate of dw/dw was significantly lower (P<0.05) than that of Dw/Dw at 2 weeks of age but not at 4 and 8 weeks of age. No significant differences in the degradation rate (K _d; %/day) were found at any age. A significant difference (P<0.05) between genotypes in the rate of synthesis (K _s; %/day) was found at 2 weeks of age (Dw/Dw=11.8, dw/dw=9.9) but not at 4 and 8 weeks of age. These results suggest that the dw gene has a depressing effect on the synthesis rate of muscle protein, and the difference between genotypes in the growth rate at the early stage is a reflection of this effect.     

Degradation of the 32-kilodalton thylakoid-membrane polypeptide of Chlamydomonas reinhardi Y-1

Isolated thylakoid membranes of Chlamydomonas reinhardi Y-1 with the 32-kDa polypeptide either radioactively labelled or unlabelled were incubated in vitro under various conditions in order to gain information about the degradation of the 32-kDa polypeptide. The degradation was higher at pH 6 compared with pH 7 and pH 8 and exhibited a temperature maximum between 20° C and 25° C (pH 6, pH 8). A light-dependent part of the total degradation was linearly dependent on white light of energy fluence rate between 1 and 20 mW·cm^-2 at 25° C and leveled out at higher fluence rates. The degradation in light was only slightly stimulated by ATP but was reduced by 3-(3-4-dichlorophenyl)-1,1-dimethylurea. Adenosine-5-diphosphate and heparin (2.7 mM and 200 g per 100 l, respectively) known to inhibit kinases, caused a 50% decrease in degradation indicating that a phosphorylation step is involved in degradating the 32-kDa polypeptide. Out of various inhibitors specific for different types of proteases, only those for thiol- and endoproteases showed intense effects. These results point to a proteolytic degradation of the 32-kDa polypetide by a thylakoid-membrane-bound thiol-endoprotease. Its activity yields soluble breakdown products with relative molecular masses (M_rs) of 23, 16.5, 11.3 and 10.7 kDa, and these are accumulated in the in-vitro system. Partial proteolytic digestion of thylakoids with Staphylococcus aureus V8 protease results in at least two labelled breakdown products (M_rs 23, and 16.5 kDa). It is assumed that cleaving at identical amino-acid residues of the 32-kDa polypeptide by the thylakoid-membrane-bound thiolendoprotease and the V8 protease results in these two breakdown products. They are derived from subsequent cleavage at amino-acid residues 60–242 and 60–189 according to the deduced protein sequence (Erickson et al. 1984, EMBO J. 3, 2753–2762).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - LDS-PAGE lithiumdodecyl sulphate-polyacrylamide gel electrophoresis - M apparent molecular mass - PSII photosystem II - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Batch xylitol production by<Emphasis Type="Italic"> Candida guilliermondii</Emphasis> FTI 20037 from sugarcane bagasse hemicellulosic hydrolyzate at controlled pH values

Batch fermentation of sugarcane bagasse hemicellulosic hydrolyzate by the yeast Candida guilliermondii FTI 20037 was performed using controlled pH values (3.5, 5.5, 7.5). The maximum values of xylitol volumetric productivity (Q _p=0.76 g/l h) and xylose volumetric consumption (Q _s=1.19 g/l h) were attained at pH 5.5. At pH 3.5 and 7.5 the Q _p value decreased by 66 and 72%, respectively. Independently of the pH value, Y _x/s decreased with the increase in Y _p/s suggesting that the xylitol bioconversion improves when the cellular growth is limited. At the highest pH value (7.5), the maximum specific xylitol production value was the lowest (q _pmax=0.085 g/l h.), indicating that the xylose metabolism of the yeast was diverted from xylitol formation to cell growth.List of symbols P _max xylitol concentration (g/l) - Q _x volumetric cell production rate (g/l h) - Q _s volumetric xylose uptake rate (g/l h) - Q _p volumetric xylitol production rate (g/l h) - q _pmax specific xylitol production (g/g h) - q _smax specific xylose uptake rate (g/g h) - _max specific cell growth rate (h^–1) - Y _p/s xylitol yield coefficient, g xylitol per g xylose consumed (g/g) - Y _p/x xylitol yield coefficient, g xylitol per g dry cell mass produced (g/g) - Y _x/s cell yield coefficient, g dry cell mass per g xylose consumed (g/g) - _cell percentage of the cell yield from the theoretical value (%) - _xylitol percentage of xylitol yield from the theoretical value (%)     

Degradation of α-amylase fromAspergillus oryzae with firmly bound proteinases at pH 3.0

Incubation of highly purified -amylase fromAspergillus oryzae (EC 3.2.1.1) with 0.01M acetate buffer, pH 3.0, resulted in degradation of the -amylase. The molecular weight values of degradation products were 42 K, 37 K, and 28 K. Incubation of the purified -amylase in 0.02m phosphate buffer, pH 7.5, at 30°C for 17 h, however, resulted in no degradation of the -amylase molecule.Incubation of the purified -amylase with proangiotensin at pH 3.0 for 24 h resulted in cleavage of Tyr⁴-Ile⁵, His⁶-Pro⁷, Pro⁷-Phe⁸, Phe⁸-His⁹, and His⁹-Leu¹⁰. Thus, it appears that proteolytic activities firmly bound to -amylase are identical withAspergillus aspartic proteinase (EC 3.4.23.6) andAspergillus acid carboxypeptidase (EC 3.4.16.1).     

Sodium ATPase and Sodium/proton Antiporter Are Not Obligatory for Sodium Homeostasis of Enterococcus hirae at Acid pH

Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na⁺-ATPase and Na⁺/H⁺ antiporter, does not grow in high Na⁺ medium at pH above 7.5. We found that 7683 grew normally in high Na⁺ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na⁺ and K⁺ were normal in this mutant. The Na⁺ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na⁺ elimination of this bacterium at acid pH is achieved by a decrease in Na⁺ entry and a normal K⁺ uptake.     

Overproduction and purification of Lon protease fromEscherichia coli using a maltose-binding protein fusion system

Lon protease, which plays a major role in degradation of abnormal proteins inEscherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form inE. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl--d-methoxynaphthylamide) and protein (-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

The BCL-xL and ACR-1 Genes Promote Differentiation and Reduce Apoptosis in Muscle Fibers of mdx Mice

The effects of the human BCL-xL and ACR-1genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 ± 0.1 and 2.1 ± 0.3%, respectively. Introduction of the dystrophin minigene (20 g of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 ± 1.4% and 4.5 ± 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 g of each plasmid per shot), the death of CSMF decreased to 3.7 ± 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 ± 2.2%. Cotransfection with the dystrophin minigene and the BCL-xL gene at 20 g of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 ± 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 g of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 ± 0.5% and significantly reduced the proportion of dying CSMF to 0.9 ± 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xLor ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.