Teizo Katsumata

Elongation factor 1 from the silk gland of silkworm Purification and some properties of its γ subunit having EF-1b activity

Elongation factor 1 (EF-l), the factor responsible for binding aminoacyl-tRNA to ribosomes, exists in multiple forms in a variety of different eukaryotes [ 1,2] . The heavier forms (EF-1, , mol. wt > 150 000) represent aggregates of a lighter form(EF-lL, mol. wt approx. 60 000) [3--S]. On the other hand, we previously noted that silk gland EF-l(APase I) consisted of three different subunits ((Y, fl and y) [6] . Their molecular weights were estimated to be about 63 000,60 000 and 30 000, respectively. Furthermore EF-I, was resolved into two complementary factors, EF-la(APase I) and EF-lb(APase II), which were thought to correspond to prokaryotic EF-Tu and EF-Ts, respectively [7]. Similar factors were also observed in pig liver [8] , rabbit reticulocyte [9] , wheat embryo [lo] and Alfemiu salina cysts [ 1 l] . From these results it is of interest and important to compare the properties of each subunit in detail. However, for this purpose the recovery of each subunit of silk gland EF-1 was low in the former procedure [7] because only a part of it was dissociated under the undenaturing conditions. Thus, we tried to dissociate and purify each subunit under denaturing conditions. In this paper we describe the procedures for the purification and some properties of the 7 subunit which showed EF-lb(EF-TSUke) activities.

Elongation factor 1 from the silk gland of silkworm. Reconstruction of EF-1M from its subunits, EF-1a and EF-1bc.

Elongation factor 1 (EF-1) which catalyzes the binding of aminoacyl-tRNA to ribosomes, exists-in multiple forms in a variety of different eukaryotes [ 1,2]. They can be classified conventionally into three groups, EF-1, (heavy form), EF-1, (medium form) and EF-1, (light form), with molecular weights of>3X lo”,1.5 X 10’ and -5.1 X 104, respectively. Although EF1, is generally grouped into EF-l,, we distinguished EF-1, and EF-1, from each other for the further study of their functional differences. It was observed that EF-1, and EF-1, represent aggregates of EF-1, [3-51. While we reported that silk gland EF-1, and EF-1, consisted of three different subunits, EF-la (o subunit, mol. wt 51 000), EF-lb (7 subunit, mol. wt 26 000), and EF-lc (p subunit, mol. wt 46 000) [6]. More recently, it was demonstrated that EF-la and EF-lb correspond to prokaryotic EF-Tu and EF-Ts, respectively [7,8]. Similar factors were also observed in pig liver [9], Artemiu saZina [lo], rabbit reticulocyte [Ill, and wheat embryo [12]. Since EF-1, and EF-1, from these organisms are thought to consist of three different subunits, reconstructions of EF-1, and EF-1, from subunits has received much attention. As EF-lb and EF-lc were separated from each other only when a denaturant such as 8 M urea is present [8], we used mainly the complex (EF-1 bc). The present work demonstrates the reconstruction of EF-1, from its subunits, EF-la and EF-lbc, or EFla, EF1 b and EF-1 c. EF-1, reconstructed from EFla and EF-I bc showed 70-80% of the activity of native EF-1,.

Exchangeability of silk gland elongation factor 1b and pig liver elongation factor 1β in polypeptide chain elongation

Three polypeptide chain elongation factors, i.e., EF-Tu, EF-Ts and EF-G, have been purified and their properties studied extensively in prokaryotes. While in eukaryotes only two factors, i.e., EF-I and EF-2, had been knawn until EF-1 was resolved into two complementary factors, EF-la(APase I) and EF-lb (APase II), in the silk gland [I ,2]. The latter which corresponds to the smallest subunit (mol. wt 26 000) of EF-1 was found to stimulate 3 reactions: (i) EF-1 aand EF-2-dependent polyphenylalar~i~e synthesis (polymerization reaction); (ii) EF-la-descendent biIlding of [~4C~Phe-tRNA to ribosomc (binding reaction); {iii) Exchange of GDP bound to EF-1 a with exogeneous GTP (exchange reaction). From these results it was concluded that the EF-lb factor corresponds to the EF-Ts [a]. Similar factors were also prepared from pig liver [3] , Arten?h safirta f4] and rabbit reticulocyte [51. However, except for the pig liver EF-1P ~haracte~zation of their proteins and functions is obscure. Recently pig liver EF-lfi (mol. wt 30 000) has been purified to apparent homogeneity and shown to stimulate the three reactions [6] . From these results it seems most likely that the EF-I/3 factor corresponds to EF-lb. This paper describes the complete ex~han~eabili~ of the silk gland EF-lb and the pig liver EF-10 in the polymerization, binding and guanine nucleotide exchange reactions. S~~arity of the amino acid compositions of EF-I b, EF-113 and EF-Ts is also noted. 2. Materials and methods

Transfection of Sendai virus F gene cDNA with mutations at its cleavage site and HN gene cDNA into COS cells induces cell fusion

SummaryIn contrast to the wild type Sendai virus fusion protein (F), a mutated F to possess a cleavage site similar to that of virulent Newcastle disease virus F, could be cleaved by proteases present in COS cells. When mutated F and hemagglutinin-neuraminidase (HN) were coexpressed at the cell surface, syncytium formation was observed.