The activities of ribosome-degrading enzymes and of a proteinase inhibitor in Tetrahymena pyriformis during starvation

• 1.1. In starved Tetrahymena acid RNase decreased but acid proteinase and a heat-stable proteinase inhibitor both increased.
• 2.2. A greater proportion of proteinase than RNase was released from the lysosomes of growing cells by homogenization, but progressively less proteinase was released during starvation.
• 3.3. Inhibitors of protein synthesis enhanced the decrease in RNase and prevented the increase in proteinase and proteinase inhibitor. Chloroquine caused a large increase in both enzymes.
• 4.4. Proteinase and RNase may be located in different lysosomes which together participate in ribosome destruction, but this process is unlikely to be limited by the total amount of these lysosomal hydrolases.

     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

The breakdown of Tetrahymena ribosomes by lysosomal enzymes: Inhibition by cytosol

• 1.1. Extracts from Tetrahymena lysosomes contained acid RNase and proteinase. At pH 7.4 there was appreciable proteinase activity which was inhibited by a heat-stable protein present in cell sap.
• 2.2. Lysosomal enzymes rapidly converted 80S ribosomes to subunits at pH 7.4. Hydrolysis of ribosomal RNA was very slow at pH 7.4 but rapid at pH 5.0.
• 3.3. These reactions were inhibited by proteinase inhibitors and by cell sap, but the latter was relatively ineffective at pH 5.0.
• 4.4. It seems unlikely that ribosome breakdown in vivo is initiated by the release of lysosomal enzymes into the cytosol.

     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

Characterization of proteolytic activities stimulated by SDS or urea and two-dimensional gel electrophoresis of proteins from Brachionus plicatilis (Rotifera)

• 1.1. Proteinases occurring in the 27,000 g supernatant of homogenates from Brachionus plicatilis have been characterized by electrophoretic techniques.
• 2.2. Several individual proteases were detected which are active in the presence of either SDS or urea.
• 3.3. By applying this knowledge about the properties of proteases occurring in Brachionus, two-dimensional electrophoretic separations of proteins from Brachionus plicatilis are made feasible without proteolytic artifacts.

     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Construction of cDNA libraries from cultures of Armillaria mellea

• 1.1. Conditions were established for growth of mycelial cultures of Armillaria mellea such that the production of its lysine-specific proteinase was maximized. Proteinase synthesis was confirmed by immunoprecipitation.
• 2.2. Mycelia grown under these same conditions were used as a source of RNA and this RNA was translatable in a wheat germ translation system to produce proteins with M_r in the range < 10,000–> 90,000
• 3.3. Double-stranded cDNA was prepared and was inserted into the EcoR1 site of λgt10 and λgt11 using an adaptor ligation strategy. Packaging of these materials yielded large cDNA libraries. That from λgt10 contained 2.9 sx 10⁶ pfu/ml with 70% recombinants whereas that from λgt11 contained 2.2 × 10⁶ pfu/ml with 60% recombinants.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts

• 1.1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri).
• 2.2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots.
• 3.3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions.
• 4.4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

     

Characterization of two distinct latent proteinases associated with myofibrils of crucian carp (Carassius auratus cuvieri)

:

• 1.1. Enzymatic properties of two distinct proteinases tightly associated with crucian carp myofibrils were characterized.
• 2.2. These proteinases were latent but activated at 50 and 60°C, respectively.
• 3.3. The optimum pH of 50°C-proteinase was neutral-alkaline, while that of 60°C-proteinase was weak acid-neutral pH.
• 4.4. Both proteinases required more than 1% NaCl for the activity, but 50°C-proteinase was partially inhibited at higher concentrations of NaCl.
• 5.5. Both proteinases were regarded as trypsin-like proteinases belonging to a serine proteinase family, but only 60°C-proteinase was sensitive to urea, n-butanol and iso-propanol.

     

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Identification of proteolytic isozymes from Antarctic krill (Euphausia superba) in an enzymatic debrider

• 1.1. A partially purified krill extract (enzymatic debrider) intended for clinical use was electrophoretically characterized by polyacrylamide gel electrophoresis (PAGE) and by crossed immunoelectrophoresis (CIE) using polyclonal rabbit antibodies.
• 2.2. Three main types of proteolytic enzymes (serine proteinase, carboxypeptidase A and B) with mol. wts of 33,000, 28,000 and 35,000, respectively, could be separated by SDS—g-PAGE under reducing conditions.
• 3.3. Routine CIE analysis of krill samples revealed four protease-active immunoprecipitates. Two of these precipitates were associated with the proteinase activity, one with carboxypeptidase A and one with carboxypeptidase B.
• 4.4. Improving resolution of CIE by extending electrophoresis in the first dimension permitted separation of three serine proteinases of which two were isozymes (II and III) and the third one was unique (I).
• 5.5. Furthermore carboxypeptidase A could also be separated into two isozymes (AI and AII) while carboxypeptidase B still exhibited one single component.
• 6.6. Six individual immunoprecipitates were thus identified and proved to be related to the protease activity. Highly purified enzymes were used as references in CIE and tandem-CIE to establish identification of each enzyme in the krill mixture.

     

Diurnal temperature sensitivity of dairy cattle in a naturally cycling environment

• 1.1.|Vaginal temperatures of 15 Bos taurus cattle were monitored for 17 days in daily pens exposed to normal environmental fluctuations.
• 2.2.|Regressions of vaginal temperature on ambient temperature were made for each collection time in the 24 h cycle. Slopes of regressions provided an index of animal sensitivity to environmental temperature.
• 3.3.|Fluctuations in sensitivity occurred throughout the day with positive slopes excepts at 0630 h.

     

Radioimmunological quantification of C21, C19 and C18 steroids in the gonads of Locusta migratoria migratorioides R. and F.

• 1.1. Levels of progesterone, pregnenolone, testosterone, 5α-dihydrotestosterone, estrone and estradiol were measured by radioimmunoassay in purified gonadal extracts of larval and adult male and female Locusta migratoria.
• 2.2. The average steroid contents varied between less than 1 ng to more than 160 ng/g tissue.
• 3.3. Young adults were treated with precocene or ketoconazole in an attempt to influence the steroid contents in gonads.
• 4.4. Ketoconazole treatment had no effect on the steroid contents in gonads whereas precocene treatment resulted in higher contents of androgens.

     

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Organohalogen constituents of the acorn worm Ptychodera bahamensis

• 1.1. Twenty organohalogen compounds, primarily phenols, were detected in the volatile extracts of the acorn worm Ptychodera bahamensis.
• 2.2. Five chlorinated compounds, previously undescribed from acorn worms, were identified.
• 3.3. Enteropneusts can be significant contributors of halogenated organics to the marine environment.

     

Deterioration of chum salmon (Oncorhynchus keta) muscle during spawning migration—III. Changes in protein composition and protease activity of juvenile chum salmon muscle upon treatment with sex steroids

• 1.1. Changes in protein composition and protease activity of juvenile chum salmon muscle upon treatment with sex steroids were investigated.
• 2.2. A slight breeding color was observed on chum salmon following the oral administration of 17α-methyltestosterone. Sarcoplasmic protein significantly decreased, while ninhydrin-positive substances from protein-free fractions significantly increased upon treatment with 17α-methyltestosterone. Autolytic activity of the fish treated with 17α-methyltestosterone drastically increased.
• 3.3. Estradiol-17β did not significantly influence the protein composition and autolytic activity.
• 4.4. These results indicate that androgen is closely related to the deterioration of chum salmon muscle.

     

Digestive protease,lipase and amylase activities in stage i larvae of the american lobster,Homarus americanus

• 1.1. Digestive protease, lipase, and amylase of Stage I larvae of the American lobster Homarus americanus are characterized.
• 2.2. A sensitive method for detection of crustacean lipase was developed using an latroscan which combines thin-layer chromatography and flame ionization detection to quantify free fatty acids generated by lipase digestion.
• 3.3. pH optima of the three enzymes occurred at or near the pH of gastric fluid.
• 4.4. A time course study demonstrated slight increases in protease and amylase activities during the first larval stage, regardless of whether the lobsters were fed or not, whereas lipase activity was constant.

     

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