Robert T. Hersh

Inactivation of High and Low Molecular Weight Chicken Antibodies by Mercaptoethanol.

Summary Inactivation of 7S and 19S chicken antibodies was dependent on mercaptoethanol concentration and time. Papaindegraded high and low molecular weight chicken antibodies lost agglutinating ability.

Regulation of glutamine synthetase in L cells by cortisol andl-glutamine

SummaryThe glutamine synthetase system of strain L mouse cells is regulated by at least two natural substances: glutamine and a glucocorticoid hormone. In a chemically defined cell culture system, cortisol or dexamethasone promotes intracellular accumulation of the synthetase by a process which depends upon genetic transcription. The accumulation is most rapid if the hormone is added to a glutamine deficient culture medium. High concentrations of glutamine promote rapid disappearance of the enzyme by a mechanism which depends on unimpaired protein synthesis. Ways in which the two regulatory substances collaborate to influence the synthetase level and other growth-related processes of L cells in culture are discussed.

The subunit structure of formyltetrahydrofolate synthetase

The molecular weight of formyltetrahydrofolate synthetase determined by the approach to equilibrium method is 2.38 · 105 ± 0.08 · 105. In the presence of 1.5–6.0 M guanidine · hydrochloride the enzyme dissociates into subunits. In 6 M guanidine · hydrochloride the molecular weight is 5.98 · 104–6.1 · 104. The enzyme is therefore a tetramer. Dissociation of the enzyme into monomers also occurs at pH 6, pH 11, and in the absence of certain monovalent cations and multivalent anions. Results of gel filtration experiments done in the presence of the substrates of the reaction and at dilute protein concentrations show that the tetramer is the catalytically active form of the enzyme. Inactivation of the enzyme by dissociation in Tris-HCl is prevented by certain monovalent cations. The presence of 2-mercaptoethanol slows down the inactivation process. The inactivation and dissociation in Tris-HCl containing 2-mercaptoethanol could be reversed by the addition of NH4Cl. Difference spectroscopy showed that upon reactivation there is an increase in the absorption spectrum of the enzyme in the region where tryptophan, tyrosine and phenylalanine absorb, indicating that some of these residues become buried in a hydrophobic region when the subunits reassociate. The rate of increase in A291 nm is equal to the rate of return of enzymic activity. NH4+, K+ and Rb+ stimulate the activity of the enzyme 2- to 3-fold under conditions where dissociation does not occur. A plot of the activity as a function of NH4+ concentration is sigmoidal. It is suggested that the monovalent cations and multivalent anions are necessary to maintain the protein in the most active tetrameric conformation. In the absence of the cation but in the presence of multivalent anions a tetramer of a different conformation is formed. This tetramer has a higher Km for formate. When both cations and multivalent anions are removed the tetramer dissociates into monomers.

Cations and the binding of polyadenylate to cellulose

Abstract At neutral pH polyadenylate (poly(A)) binds to unmodified cellulose in high salt solution and is released in low salt solution. The salt effect is due principally to the cation, not to the ionic strength of the solution. Monovalent cations are not equally supportive of binding. The order of their effectiveness corresponds to a Hofmeister lyotropic series, increasing with decreasing radius of the hydrated cation. The cellulose · poly(A) interaction does not seem to be due to simple hydrophobic attraction but is consistent with association by π—π interaction between aromatic constituents of lignin of the cellulose and the pruine nuclei of the poly(A). Fractional elution of poly(A) from cellulose can be accomplished by gradually decreasing the salt content of the eluting buffer. The poly(A) oligomers which disengage from cellulose at rather high salt concentrations seem to be shorter than those which are released only at lower salt concentrations. This discrimination may serve as a basis for the fractionation of poly(A)-containing RNAs of which the poly(A) suffixes differ in length.

ACTION OF RIBONUCLEASE UPON THE POLYSOMES OF CULTURED MOUSE CELLS

SummaryPancreatic ribonuclease (RNase) and3H-uridine were used to study certain compositional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside,3H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase-resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomycin D inhibition of RNA synthesis was used to confirm this identification.The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10−6m) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes.