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A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.

A proteinase was purified from the culture filtrate of psychrophilic Escherichia freundii, by a combination of procedures, such as precipitation with ammonium sulfate, batchwise treatment with CM-cellulose, chromatography on DEAE-Sephadex and gel filtration on Sephadex G-100. The final enzyme preparation was homogeneous with respect to sedimentation in the ultracentrifuge, acrylamide-gel electrophoresis and immunodiffusion. The enzyme was an alkaline proteinase with the optimum pH at 10.0. The optimum temperature was 25 "C at pH 10.0. The proteinase of psychrophilic E. freundii was fairly thermolabile. The purified enzyme was inhibited by metalchelating agents but not by diisopropylphosphofluoridate and p-chloromercuribenzoate.The molecular weight of the proteinase was estimated to be 45800 by sedimentation-equilibrium studies, 45 000 by gel-filtration method and 51 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulfate. The calculation of minimum molecular weight on the basis of zinc content yielded a value near 45400 which indicated the presence of 1 g-atom zinc per mol enzyme. The proteinase had a sedimentation coefficient of 4.12 S, a diffusion coefficient of 77.4 pm2/s, a partial specific volume of 0.718 ml/g, an isoelectric point of pH 5.2, a t 280 nm of 12.45, and an intrinsic viscosity of 0.048 dl/g. The amino-acid composition indicated 429 residues per molecule and the absence of sulfhydryl groups and disulfide bridges. The NHzterminal amino acid of the enzyme was shown to be glycine.The extracellular Escherichia proteinase has been found to be a zinc-containing alkaline metallo-proteinase.Psychrophilic microorganisms are capable of growing a t 0 "C and lower temperatures, compared to a minimum of about 10 "C for mesophilic. It has been lu~own that many enzymes obtained from such psychrophilic microorganisms are thermolabile and have a low activation energy. I n order to determine whether psychrophilicity of such an enzyme results from a characteristic molecular structure or others, the physicochemical properties of the enzyme should be studied in detail. Nunokawa and McDonald [l] and Kato et al.[2] reported the purification and some properties of the proteinases from an obligately psychrophilic red-pigmented bacterium and a psychrophilic Pseudmnonas strain, respectively, but their physicochemical properties are not described.We have discovered that a psychrophilic strain of Escherichia freundii isolated from soil produced a rich amount of extracellular proteinase [3]. Formation of extracellular proteinases by the genus Escherichia has not been reported before. Among the family The present paper is concerned with the purification and enzymatic and physicochemical properties of an extracellular proteinase of a psychrophilic strain of E . freundii. This study was undertaken to elucidate the mechanism of psychrophilicity of psychrophilic enzyme and in order to compare the properties of an Escherichia proteinase with those of the well-characterized Serratia proteinase [5-71. UT...

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Proteolytic enzymes derived from thermophilic streptomyces sp. strain 1689 were purified and some properties were studied. A 8-fold purification was obtained from the culture supernatant by ammonium sulfate fractionation, acetone precipitation, and chromatography on CM-Sephadex. Two proteinases of almost identical properties were fractionated on CM-Sephadex chromatography. The purified preparations appeared to be homogeneous on ultracentrifugation. The optimum pH for proteolytic activity on casein was found to be pH 10.610.8. The stability was considerably increased by the addition of Ca++, and the proteinases exhibited a relatively high thermal stability. Enzyme activity was inhibited by oxidizing agents, PCMB, potato inhibitor, DFP, and heavy metal ions. Na+ K+ Mg++ and Fe++ showed an activating effect.