Measurement of protein concentration with interferences optics

Babul, Jorge; Stellwagen, Earle

doi:10.1016/0003-2697(69)90172-9

Cited by 399 publications

(165 citation statements)

References 16 publications

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“…Protein concentrations were determined in the ultracentrifuge employing the Rayleigh interference optical system assuming 41 fringes equivalent to 10 mg/ml [10]. For CD and sedimentation equilibrium studies the protein was initially dissolved in 10 -4 N HC1 and then dialysed against the buffer.…”

Section: Methodsmentioning

confidence: 99%

Separation and characterization of the 37 000 dalton component of the troponin system

1974

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Section: Methodsmentioning

confidence: 99%

Separation and characterization of the 37 000 dalton component of the troponin system

1974

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“…cm-'. This value was determined using interference optics in the analytical ultracentrifuge according to the procedure of Babul and Stellwagen [88]. Actin concentration was determined by absorbance at 290 nm using an absorption coefficient of 0.63-mg .…”

Section: Preparation Of Proteinsmentioning

confidence: 99%

Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides

Trayer

1983

European Journal of Biochemistry

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The unique fast-reacting cysteine residue (SH,) of myosin subfragment 1 (Sl), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N-(bromoacety1)-N'-(1-sulpho-5-naphthy1)ethylenediamine (1, and 5-(iodoacetamido)fluorescein (5-IAF), whose spectral properties render them a particularly effective donor-acceptor pair in fluorescence energy transfer studies. The transfer efficiency of [40][41][42][43][44][45] represented a spatial separation of the chromophores of about 5 nm, which is in reasonable agreement with the value of 6 nm reported earlier for similarly labelled S1, prepared by papain digestion, and actin [Takashi, R. (1979) Biochemistry, 18, 5164-51691. This transfer efficiency did not change when the doubly-labelled binary complex was formed: (1) with acto-Sl(A1) or acto-Sl(A2) at 10-200 mM KCl, pH 7-8 and different buffer conditions; (2) with either S1 isoenzyme and regulated actin (i.e. actin with tropomyosin and troponin) both in the presence and absence of Ca2+ or when the donor and acceptor attachment sites were reversed. Analysis of donor and acceptor polarized fluorescence showed that the chromophores are not randomly orientated (i.e. x2 # 2/3), but they do have some motion relative to either protein.From a knowledge of the limiting values for x2, the intersite distance for donor and acceptor chromophores was calculated to be in the range 3.9-6.7 nm.Addition of MgATP to the doubly-labelled acto-S1 complex eliminated energy transfer but this was recovered when ATP hydrolysis was completed. By utilizing the known binding constants between S1, actin and either MgADP or MgAdoPP Biol. Chem. 255,, the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto-S1-nucleotide complex (z 50%) and minimise the amount of binary complex ( 5 2%). The spatial separation of the chromophore interaction sites in the ternary complex was found to be the same with both nucleotides and indistinguishable from that found with the binary complex. A similar strategy was employed to compare the conformations of the binary and ternary complexes by 'H-NMR spectroscopy. In these experiments about 90 % of the S1 was in the form of the ternary complex. There was no noticeable change in the acto-S1 spectra upon addition of either MgAdoPP[NH]P or MgADP. These observations support the conclusion that there is no large change in structure in the 'rigor' binary acto-S1 complex when it binds either ADP or AdoPP[NH]P.The interaction between actin and myosin is central to the energy transduction process in muscle. However, whereas actin is a highly conserved protein [I], the subunit of the hexameric myosin molecule can differ widely both between tissues and within a single tissue [2]. It is thought that this variation in the sequence and composition of the myosin subunits is largely responsible for the different contractile properties exhibited by different muscle (and non-muscle) tiss...

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“…The values obtained either by this procedure or by using the absorbance index, A:$,,,,,, of 12.4 for phosphorylase kinase [6], were within 5 % of each other. The concentration of the 6 subunit was also determined by the Lowry procedure, and the validity of this method was established by refractometric measurements in the analytical ultracentrifuge [6,29]. A solution of 6 subunit which yielded a protein concentration of 1.9 mg/ml by the Lowry procedure possessed 8.1 interference fringes, corresponding to a protein concentration of 2.0 mg/ml [6,29].…”

Section: Enzyme Assaysmentioning

confidence: 99%

The Role of Calmodulin in the Structure and Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle

et al. 1979

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Phosphorylase kinase from rabbit skeletal muscle has been shown to possess the structure ( a j y 6)4, where the b-subunit is identical to the calcium binding protein, termed calmodulin. The amount of calmodulin was found to be stoichiometric with the M., p and y subunits in all preparations of phosphorylase kinase, and was not dissociated from the enzyme even in the presence of 8 M urea, provided that calcium ions were present.The activity of phosphorylase kinase was increased by the addition of calmodulin to the assay, and half-maximal activation was observed at a molar ratio, calmodulin/phosphorylase kinase, of 20 : 1. At saturating concentrations of calmodulin, all preparations of phosphorylase kinase had a specific activity of 13.5 t-1.0 U/mg at pH 8.2. In the absence of calmodulin, the specific activity ranged from 2 -7 U/mg, so that the stimulation by calmodulin varied from 2 -7-fold with different preparations of phosphorylase kinase. The molecular basis for this variability is discussed.The stimulation of the activity by calmodulin was prevented by the addition of troponin-I or the antipsychotic drug trifluoperazine, whereas these compounds had little effect on the calciumdependent activity in the absence of calmodulin. These results demonstrated that the activation by calmodulin was caused by the interaction of a second molecule of calmodulin with phosphorylase kinase. The existence of an additional calmodulin binding site was also indicated by the finding that phosphorylase kinase bound to calmodulin-Sepharose in the presence of calcium ions.Experiments with ICR/IAn mice which lack muscle phosphorylase kinase activity and C3H/He-mg mice with normal activity, demonstrated that when muscle extracts were fractionated with ammonium sulphate, 90 -95 % of the calmodulin which precipitated at 0 -35 % ammonium sulphate was bound to phosphorylase kinase. This information was used to show that at least 35 % of the calmodulin in low ionic strength EDTA extracts of rabbit skeletal muscle was bound to phosphorylase kinase.Muscle extracts from ICR/IAn mice and C3H/He-mg mice had identical myosin light-chain kinase activities, showing that there was not a generalized defect in calmodulin-dependent enzymes in ICR/IAn mice. The skeletal muscle extracts of ICR/IAn mice contained 60% of the calmodulin found in C3H/He-mg mice, and addition of calmodulin did not restore phosphorylase kinase activity to muscle extracts prepared from ICR/IAn mice. This indicated that the lack of phosphorylase kinase activity was not caused by an absence of the calmodulin molecule.The role of calmodulin in the regulation of muscle phosphorylase kinase activity is discussed.Phosphorylase kinase catalyses the phosphorphosphorylation, catalysed by cyclic-AMP-dependent ylation and activation of glycogen phosphorylase protein kinase [4]. and thereby stimulates the rate of glycogenolysis.Several years ago, phosphorylase kinase was The activity of phosphorylase kinase from rabbit found to contain three types of subunit [5,6] and to skeletal muscle ...

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Measurement of protein concentration with interferences optics

Cited by 399 publications

References 16 publications

Separation and characterization of the 37 000 dalton component of the troponin system

Separation and characterization of the 37 000 dalton component of the troponin system

Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides

The Role of Calmodulin in the Structure and Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle

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