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R Dubrow

Publications and source records attributed to R Dubrow.

8 recordsLinked to original sources
PubMed1979

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons. This protein also showed the greatest change in synthesis during the transit from G0 to S phase. Its synthesis rose to a maximum 4--6 hr after stimulation and then declined as cells entered S phase. The protein was present in both nuclear and cytoplasmic extracts. It was identified as actin on the basis of its mobility on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels. Other proteins synthesized preferentially by stimulated cells had molecular masses of 57,000 daltons (cytoplasmic), 33,000 daltons (cytoplasmic and nuclear), and 15,000 daltons (nuclear). The synthesis of the 57,000 and 33,000 dalton proteins increased gradually after stimulation and remained high during S phase. The 15,000 dalton proteins began to be synthesized as cells entered S phase. The preferential synthesis of these proteins provides biochemical markers for the transition from quiescence to proliferation.

Actins↗
PubMed1978

2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines.

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2-amino-isobutyric acid and 3-O-methyl-D-glucose were measured in sparse, confluent, serum-starved, and serum-stimulated cultures. As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101. The density revertant F1SV101, which has regained density regulation of growth similar to 3T3 cells, has also regained density regulation of transport. Neither growth nor transport were serum dependent. The serum revertants AgammaSV7 and LsSV6 have regained both density and serum regulation of growth, but not according to the original mechanism of 3T3 cells of entry into a Go state. Transport was high under conditions of confluence or serum deprivation. Thus for these cells rates of transport were not reduced simply as a consequences of slower cell growth nor were low transport rates responsible for growth arrest. The data are consistent with the possibility that growth arrest specifically in the G0 state could shut off a number of cellular activities, including transport.

Aminoisobutyric Acids↗
PubMed1977

A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH.

The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5.

Alcohol Oxidoreductases↗
PubMed1977

Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase.

The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P. These studies led to the scheme below for the ternary complex interconversion. E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations. Step 3 is the hydride transfer. Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values. At lower pH values, the hydride transfer step is also partially rate-determining. The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values. At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step. Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion. Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme. When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred. This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.

Alcohol Oxidoreductases↗
PubMed1977

Transient kinetic studies on the allosteric transition of phosphoglycerate dehydrogenase.

Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine. The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration. At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1. The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete. These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme. A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state. An alternative model, which we favor, proposes two classes of inhibitor binding sites. The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I. (1968) J. Biol. Chem. 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration. At pH 8.5, kobs,f.q. ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations. These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme. Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm. A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex. The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I. (1977) J. Biol. Chem. 252, 1539-1551). The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j. This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity. During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate. kobs,j and kobs,f.q. were also measured using glycine, another inhibitor. The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.

Alcohol Oxidoreductases↗