Jorge J. Sánchez

Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease.

A serine protease was purified from Natronococcus occultus stationary phase culture medium (328‐fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin‐like activity, is stable and active in a broad pH range (5.5–12), is rather thermophilic (optimal activity at 60 °C in 1–2 m NaCl) and is dependent on high salt concentrations for activity and stability (1–2 m NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross‐reactivity with culture medium from other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests that Natronococcus occultus extracellular protease may be a novel enzyme.

Myofibril-bound serine protease and its endogenous inhibitor in mouse: extraction, partial characterization and effect on myofibrils.

The protein content of muscle is determined by the relative rates of synthesis and degradation. The balance between this process determines the number of functional contractile units within each muscle cell. Myofibril-bound protease, protease M previously reported in mouse skeletal muscle could be solubilized from the myofibrillar fraction by salt and acid treatment and partially purified by Mono Q and Superose 12 chromatography. Isolated protease M activity in vitro on whole myofibrils resulted in myosin, actin, troponin T, alpha-actinin and tropomyosin degradation. Protease M is serine type and was able to hydrolyze trypsin-type synthetic substrates but not those of chymotrypsin type. In gel filtration chromatography, protease M showed Mr 120.0 kDa. The endogenous inhibitor (MHPI) is a glycoprotein (110.0 kDa) that efficiently blocks the protease M-dependent proteolysis of myofibrillar proteins in a dose-dependent way, as shown by electrophoretic analysis and synthetic substrates assays. Protease M-Inhibitor system would be implicated in myofibrillar proteins turnover.

Purification and characterization of a proteinase inhibitor from white croaker skeletal muscle (Micropogon opercularis)

A trypsin proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Micropogon opercularis). Previously, we had described the occurrence in fish muscle of a serine protease (proteinase I) which showed a great capacity to degrade whole myofibrils in vitro and an endogenous inhibitor that prevented the action of the protease, both on natural and artificial substrates. In this paper, we report the purification and further biochemical characterization of the endogenous trypsin inhibitor. The purification was carried out by DEAE-Sephacel, Con A-Sepharose, Sephacryl S-300 and Mono Q. Throughout the purification procedure, trypsin inhibitory activity was assayed using azocasein as substrate. The molecular mass of the inhibitor was 65 kDa, as estimated by SDS-PAGE and gel filtration. The trypsin inhibitor is a glycoprotein, as deduced by the fact that it binds to Con A-Sepharose and stains with PAS and showed a wide range of pH stability (from 5 to 11). The thermal stability of the inhibitor considerably decreased at temperatures >60 degrees C. Assays of the inhibitor against various proteases indicated that it is highly specific for serine proteases, since it did not inhibit proteases belonging to any other groups. The inhibitor was able to inhibit the endogenous target enzyme (proteinase I) in a dose-dependent manner, with a 50% inhibition at a molar ratio close to 1. The present work contributes to improving our understanding of the physiological role of the proteinase I-inhibitor system in muscle protein breakdown, as well as its influence on post mortem proteolysis.

Fructokinase from rat liver. I. Purification and properties

Abstract Fructokinase (ATP: d -fructose-1-phosphate transferase, EC 2.7.1.3) from rat liver has been purified 400-fold. The purification procedure involves an acid treatment, a heat step at 65°, (NH4)2SO4 fractionation, chromatography on Sephadex G-100 and finally (NH4)2SO4 extraction. The enzyme appears nearly homogenous by density gradient centrifugation but gives a single peak in sedimentation velocity analysis. Purified liver fructokinase has a K m of 0.46–0.80 mM for fructose and 1.56–1.33 mM for MgATP at a K+ concentration of 0.4 and 0.1 M, respectively. The enzyme also phosphorylates l -sorbose and d -tagatose. No difference could be found in the phosphorylation of the pyranose and furanose forms of fructose. The enzyme is inhibited by p- chloromercuribenzoate and is stable up to 50–55°.

Autoproteolytic activation of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease.

The haloalkaliphilic archaeon Natronococcus occultus produces an extracellular serine protease in the stationary growth phase and upon starvation. Two proteins immunologically related to the extracellular protease were detected into the cells: P200 and P190. P200 was detected at early stages of growth and its relative amount decreased as the culture reached the stationary growth phase, concomitantly with the appearance of P190 and proteolytic activity, suggesting that P200 may be the precursor of the secreted protease and P190 the mature enzyme. Both proteins were also detected in the culture medium. Conversion of inactive P200 into active P190 was attained in cell‐free culture medium from stationary phase but not from exponential phase. This process was prevented in the presence of PMSF and could be attained by addition of purified mature extracellular protease to P200. Altogether these results indicate that activation of Natronococcus occultus extracellular protease may be autoproteolytic and that factor/s present in stationary phase culture medium may be required for this process.

A cysteine protease from myxosporean degrades host myofibrils in vitro

Abstract Pseudocysts of Kudoa rosenbuschi (Myxozoa, Myxosporea) contain a cysteine protease (Mr 147.2 kDa) which has a great ability to degrade in vitro constituents of whole myofibrils of the Atlantic hake, Merluccius hubbsi , skeletal muscle. The enzyme was purified to apparent homogeneity: a single peak of azocaseinolytic activity was observed after anion exchange chromatography and gel filtration. The electrophoretic patterns of this protease under non-denaturing conditions and gelatin substrate gels show a single band while the electrophoretic pattern under denaturing conditions shows a band corresponding to 25.5 kDa. The activity of the purified enzyme was maximum in the broad pH range between 4 and 7. Cysteine protease inhibitors were able to reduce the enzyme activity whereas specific inhibitors of serine proteases and aspartic proteases were ineffective. A weak activation was observed with EDTA and with 2-mercaptoethanol. The enzyme degrades M. hubbsi myofibrillar proteins, mainly titin and myosin heavy chain, at 37°C, in vitro. No degradative activity was detected on sarcoplasmic proteins. This enzyme is likely to be responsible for the proteolysis previously observed in skeletal muscle around white and black pseudocysts.

Purification and characterization of a latent form of multicalytic proteinase from fish muscle

1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle.

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.

Protease I Inhibitor System in Fish Muscle: A Comparative Study

Abstract A protease and a trypsin inhibitor were partially purified from skeletal muscle of fishes phylogenetically different. The protease fraction from each fish species studied was immunologically similar to protease I from white croaker. Each endogenous inhibitor fraction inhibited protease I fraction from other fish species. The present results suggest that protease I inhibitor system is present in all fish species studied.

Fructokinase from rat liver. II. The role of K+ on the enzyme activity

Abstract The effect of K + on the activation of fructokinase (ATP: d -fructose- i -phosphate transferase EC 2.7.1.3) from rat liver has been studied. Substrate (fructose) kinetics are Michaelian in the presence of K + , but K + stimulates the reaction in a cooperative manner. ADP is a noncompetitive inhibitor towards frutose and competitive with respect to MgATP. K + partially reverts ADP inhibition. In a purely kinetic sense, ADP and K + are competitive antagonists. Fructokinase activity is dependent on the ratios ATP 4− /MgATP 2− and KATP 3− /MgATP 2− . The optimal ratio varies depending on the enzyme saturation and the K + concentration. Slight modification in the level of ATP 4− or Mg 2+ alter the optimal ratio, provoking inhibition by either ATP 4− or Mg 2+ . K + can partially revert this inhibition.