Separation of three microbial amino acid polymerization factors.

Abstract

Two fractions which complement the ribosomes for amino acid polymerization were obtained by DEAE-Sephadex chromatography of E. coli extracts.'-3 Using a stepwise procedure,' the fraction eluting first was heat-stable; the more retarded fraction, which coincided with a ribosome-dependent GTPase,2 3 was heat-labile. However, when a linear salt gradient was employed for elution,3 it was the earlier fraction that turned out to be unstable and the later stable.In an attempt to solve the paradox of this switch in stability, the polymerizing factors were isolated from Pseudomonas fluorescens. A different microorganism was chosen with the hope that new facets of the problem of protein synthesis might be exposed, and that some possible advantages in the stability or ease of isolation of the factors might be observed. Using Ps. fluorescens and E. coli factors interchangeably, it was found that the T fraction3 could be split into a stable component, T8, and an unstable component, Tu.4 Tu is very heat-labile and, depending on the elution procedure, can appear associated with the earlier or the later DEAE-Sephadex eluates.Materials and Methods.-Pseudomonas fluorescens, NCIB 8248, was grown on a synthetic medium as described by Wade and Robinson.6 The cells were harvested in log phase, washed twice with 0.01 M Tris-HCl buffer, pH 7.4, and 0.01 M MgCl2, and stored at -15°as a paste. E. coli B was grown as described.6 E. coli soluble RNA (General Biochemicals, Chagrin Falls, Ohio) was charged with radioactive phenylalanine according to Conway,7 using 0.6 JAmole of C'4-L-phenylalanine, specific activity 351 gc/,gmole. The specific activity of the charged sRNA was 0.46 ,gmole C'4-Lphenylalanine per ,ug sRNA.Preparation of ribosomes: In most of the experiments, E. coli ribosomes were employed because they were more stable to extensive salt washes than those from Ps. fluorescens; otherwise, the ribosomes were completely interchangeable.Ribosomes from E. coli B were isolated by a modification of the method of Nishizuka and Lipmann.3 After sedimentation at 105,000 X g, they were washed in 0.01 M Tris-HCl buffer, pH 7.4, 0.5 M NH4Cl, and 1 X 10-4 M MgCl2. In the subsequent washes, the magnesium coneentration was gradually increased to 1 X 10-3 M in the second, to 0.5 X 10-2 M in the third, and to 1 X 10-2 M in the fourth wash. In the final two washes, the magnesium concentration was maintained but the 0.5 M NH4Cl was omitted. Ribosomes thus isolated were suspended in the buffer mixture of the last wash to give 100 mg/ml. One mg dry weight of the ribosomes is assumed to be equivalent to 14.4 OD units at 260 m1A. This preparation is stable for at least 9 months at -15°; it is free of T. and Tu, but retains a small amount of G (5-8 jujAmoles of p32. phosphate were liberated from GTP32 per 100 gg of ribosomes per 10 min at 300, assayed as described3).For the assay of T. activity, Ps. fluorescens ribosomes, sedimented through 10% sucrose, have proved very useful. They retain an excess of G (1.5 msmoles of P32-phosphate were liberated from G...

Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins

“…The existence of a stable complex between Ras2p and Sdc25p-C was suggested by earlier work with other GTP-binding proteins, such as elongation factor Tu (Lucas- Lenard and Lipmann, 1966), smg p21 (Mizuno et al, 1991), and ran-TC4 (Bishoff andPonstingl, 1991a and1991b), and their specific GDP/GTP exchange factors. The observation (Poullet, P., unpublished results) that Sdc25p-C stabilizes for several days the activity of the otherwise very unstable nucleotide-free ras proteins, indicated a tight interaction between these two proteins.…”

Protection of E. coli ribosomes against colicin E3‐induced inactivation by bound aminoacyl‐tRNA

“…The ribosomes were then suspended in and dialyzed against buffer A containing 0.1 M NH,Cl, 0.01 M Mg(OAc),, 0.05 M Tris-HCl (pH 7.5) and 0.01 M mercaptoethanol. EF-T (Tu+Ts) was prepared according to Lucas-Lenard and Lipmann [7].…”

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

“…EF-Tu of E. coli is known to be highly sensitive to thermal inactivation [13]. This protein heated at 60°C for 5 min lost its ability to bind GDP ( fig.5A).…”