Japan

Lennon, Mark M.

doi:10.4018/9781591403159.ch009

Cited by 32 publications

(58 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The concentrations of Fru(l,6)P2 required to activate the system maximally in the presence of dithiothreitol were much lower than when this compound was added alone (Fig. 7 and Table4) and are closer to the physiologically relevant levels likely to be found in untreated lysates [18]. d ) Miscellaneous compouids.…”

Section: Cornpouiids Which Pseverzt Early Shut-qfl Of' Psotein Sjmthesismentioning

confidence: 74%

The Preparation and Properties of Gel-Filtered Rabbit-Reticulocyte Lysate Protein-Synthesis Systems

et al. 1983

View full text Add to dashboard Cite

When rabbit reticulocyte lysates were gel-filtered on Sephadex (3-25 or (3-50, the rate of protein synthesis in the gel-filtered lysate was the same as in the parent lysate provided appropriate concentrations of ATP, G T P and spermidine or spermine were added. The polyamines were found to increase the rate of synthesis and to lower the Mg2 + optimum. Although gel-filtered lysates prepared in this way syntheses protein at a high initial rate, this rate is maintained for only about 20 min, after which it declines rapidly to a very low rate, even though optimal concentrations of haemin are present. This shut-off was completely prevented by the addition of low concentrations of sugar phosphates capable of acting as NADPH generators: glucose 6-phosphate, 2-deoxyglucose 6-phosphate, ribulose 5-phosphate and ribose 5-phosphate. Although the addition of isocitrate resulted in the generation of NADPH, the stimulation of protein synthesis was normally less than was achieved with glucose 6-phosphate. Dithiothreitol also promoted only partial activation, as did fructose 1,6-bisphosphate, a sugar phosphate which does not generate NADPH in this system. Full stimulation was observed, however, when both fructose 1,6-bisphosphate and either dithiothreitol or isocitrate were added. It is argued that the maintenance of maximum rates of protein synthesis in gel-filtered lysates requires the presence of both a sugar phosphate and a reducing agent, which can be dithiothreitol or an NADPH-generating system. Low concentrations of thioredoxin were shown to be required for the stimulatory effect of NADPH-generating systems, but not for stimulation by dithiothreitol. On the basis of these studies, a recommended procedure is given for the preparation of highly active gel-filtered lysates and gel-filtered nuclease-treated lysates.The rabbit reticulocyte lysate is unique amongst mammalian cell-free systems in that it synthesises protein at the same rate as the intact cell for periods of 60 min or more [I]. Almost all the work that has been done with this system has used crude undialysed lysates for the reason that dialysis or gel-filtration generally results in very considerable loss of translation activity. For many years this has been a challenging problem, since the ability to make gel-filtered lysates as active as the parent lysate would not only extend the utility of the lysate as a system for translation of heterologous mRNA [2], but would also allow one to define what low-molecular-weight cofactors are required to maintain a protein synthesis rate comparable to that in the intact cell. In fact, we and others have discovered some conditions which allow gel-filtered lysates to synthesise protein at high rates [3-91, but the exact nature of the requirements for low-molecular-weight cofactors, and the reasons underlying these requirements have remained obscure and controversial. In this and the following papers we describe recent work which clarifies and largely solves these outstanding problems.Although the data are extensive and the ar...

show abstract

Section: Cornpouiids Which Pseverzt Early Shut-qfl Of' Psotein Sjmthesismentioning

confidence: 74%

The Preparation and Properties of Gel-Filtered Rabbit-Reticulocyte Lysate Protein-Synthesis Systems

et al. 1983

View full text Add to dashboard Cite

show abstract

“…the 2s 2 2p 2 3 P, 1 D, 1 S and 2s2p 3 5 S levels. The previous work of Lennon & Burke (1994) provides data for electron temperatures in the range log T e (K) = 3.0-5.0 for these 13 transitions. Stafford et al (1994) provide effective collision strengths for these transitions for temperatures in the range log T e (K) = 3.7-5.1.…”

Section: R E S U Lt S a N D Discussionmentioning

confidence: 99%

“…Previous calculations for the effective collision strengths among the N II levels include two R-matrix calculations both carried out in 1994. Lennon & Burke (1994) carried out a 12-state calculation, the targets being of the form 2s 2 2p 2 , 2s2p 3 and 2p 4 . Three real orbitals (1s, 2s, 2p) and four pseudo-orbitals (3s, 3p, 3d, 4s) were included, and the effective collision strengths were produced over the electron temperature range of log T e (K) = 3.0−5.0 (i.e.…”

Section: Introductionmentioning

confidence: 99%

Effective collision strengths for transitions in singly ionized nitrogen

Hudson

Bell

2004

Monthly Notices of the Royal Astronomical Society

View full text Add to dashboard Cite

The R‐matrix method has been used to calculate effective collision strengths for the electron‐impact excitation of the carbon‐like ion N ii. The lowest 23 LS target states are included in the expansion of the total wavefunction, consisting of the nine n= 2 states with configurations 2s22p2 and 2s2p3, along with 14 n= 3 states with configuration 2s22p3l (l= s,p,d). The fine‐structure collision strengths have been obtained by transforming to a jj‐coupling scheme using Saraph's jajom program, and have been determined at a sufficiently fine energy mesh to delineate properly the resonance structure. Effective collision strengths are obtained by averaging the electron collision strengths over a Maxwellian distribution of velocities. Results are presented for transitions among the 2s22p23P, 1D, 1S and 2s2p35S levels for electron temperatures in the range log Te(K) = 2.0−5.1, appropriate for astrophysical applications.

show abstract

“…Chemicals were from Sigma or Merck. Clones R14208, N23530, N42133, W05752, H38879 and T82144 from the IMAGE consortium [7], were kindly provided by the UK HGMP Resource Centre.…”

Section: Methodsmentioning

confidence: 99%

Human l‐3‐phosphoserine phosphatase: sequence, expression and evidence for a phosphoenzyme intermediate

et al. 1997

View full text Add to dashboard Cite

We report the sequence of the cDNA encoding human L-3-phosphoserine phosphatase. The encoded polypeptide contains 225 residues and shows 30% sequence identity with the Escherichia coli enzyme. The human protein was expressed in a bacterial expression system and purified. Similar to known L-3-phosphoserine phosphatases, it catalyzed the Mg 2+ -dependent hydrolysis of L-phosphoserine and an exchange reaction between L-serine and L-phosphoserine. In addition we found that the enzyme was phosphorylated upon incubation with L-| 32 P|phosphoserine, which indicates that the reaction mechanism proceeds via the formation of a phosphoryl-enzyme intermediate. The sensitivity of the phosphoryl-enzyme to alkali and to hydroxylamine suggests that an aspartyl-or a glutamylphosphate was formed. The nucleotide sequence of the cDNA described in this article has been deposited in the EMBL data base under accession number Y10275.

show abstract

Japan

Cited by 32 publications

References 0 publications

The Preparation and Properties of Gel-Filtered Rabbit-Reticulocyte Lysate Protein-Synthesis Systems

The Preparation and Properties of Gel-Filtered Rabbit-Reticulocyte Lysate Protein-Synthesis Systems

Effective collision strengths for transitions in singly ionized nitrogen

Human l‐3‐phosphoserine phosphatase: sequence, expression and evidence for a phosphoenzyme intermediate

Contact Info

Product

Resources

About