The shape of protein S4 from Escherichia coli ribosomes in solution was determined by hydrodynamic methods and low-angle X-ray scattering. The molecular weight of 24000 determined by low-angle X-ray scattering is within 3 % of that found by sedimentation equilibrium analysis and 8 of that determined by amino acid sequence work. The radius of gyration of 3.36 nm. the radius of gyration of the cross section of 0.41 nm and the hydrodynamic studies revealed that protein S4 is not spherical, but rather has a markedly extended shape. Calculations of different conformations, e.g. random coil, based on the parameters evaluated from hydrodynamic methods, revealed a rod-like structure of S4 with a length of 14 nm and a diameter of 1 nm. This is supported by a model of an equivalent scattering particle of uniform density based on all parameters obtained in this study.Physiochemical characterizations of ribosomal proteins, especially those which specifically bind to 23-S, 16-S or 5-S RNA, are of importance for studies on protein-RNA interactions and on the topography of the ribosoinal particle. Although the chemical and immunological properties of the isolated proteins [l -51 have been studied intensively, very little is known about the shape of the individual proteins, e.g. if they are globular or highly asymmetric. Only the shapes of the proteins L7 and L12 have recently been determined [6].Protein S4, of which the primary sequence has been determined [7], is one of the most interesting proteins in the Escherichiu coli 30-S ribosomal subunits. It binds specifically to the 16-S RNA [8], and details of the interaction between proteins S4 and 16-S RNA have been studied [9-131. Furthermore, mutants with drastically altered S4 proteins, whose binding to 16-S RNA can be impaired, have been investigated [14-211. Information on the shape of protein S4 would help to understand its role in protein-RNA interactions.In the present study attempts were made to determine the shape of protein S4 in solution, e . g . reconstitution buffer [22]. The gross conformation of the S4 molecule in solution was determined by different techniques, of which low-angle X-ray scattering proved to be the most useful. Moreover, several hydrodynamic methods revealed that S4 has a markedly extended shape. MATERIALS AND METHODS Isolation of Protein S4

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.

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Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.