Crystalline Fraction I Protein: Preparation in Large Yield

Chan, P. H.; Sakano, Katsuhiro; Singh, Shalini; Wildman, S. G.

doi:10.1126/science.176.4039.1145

Cited by 132 publications

(37 citation statements)

References 3 publications

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“…Although attempts to crystallize barley Fraction I using the method developed for the tobacco protein by CHAN et al (7) met with no success, it was possible to obtain crystalline barley Fraction I protein by the ammonium sulphate method of JAKOBV (17). The crystals obtained using this technique had exactly the same morphology as crystalline tobacco Fraction I prepared in this laboratory and as described by KAWASHIMA and WILD-MAN (20).…”

Section: Crystallizationmentioning

confidence: 68%

“…The method has also been used with both tobacco and spinach leaves and has been found equally suitable for this material. Fraction I of tobacco prepared by our method is identical to three times crystallized protein prepared by the method of CHAN et al (7) as judged by the criteria of size, amino acid composition and behavior on SDS gels.…”

Section: Smallmentioning

confidence: 99%

“…Fraction I protein from tobacco was crystallized by the method of CHAN et al (7). Fraction I protein from barley was precipitated in 55 % saturated ammonium sulphate, and the precipitate progressively resuspended in 54 %, 53 % down to 42 % saturated ammonium sulphate until crystals were observed in the supernatant after centrifugation at 1000 )< g for 5 minutes (17).…”

Section: Protein Crystallizationmentioning

confidence: 99%

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Ribulose-1,5-diphosphate carboxylase from barley (Hordeum vulgare)

Strøbæk

Gibbons

1976

Carlsberg Res. Commun.

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This paper presents a simple and rapid procedure for the isolation of ribulose-l,5-diphosphate carboxylase from leaves. By column chromatography on Sephadex G-25 and Sepharose 6B combined with concentration by ultrafiltration high yields of undenatured protein are obtainable in less than one working day. RudP carboxylase purified from barley in this way has been characterized. The molecular size is similar to that of spinach, wheat and oat. The apparent molecular weight determined by column chromatography was found to be 510,000, approximately 3 % higher than that for the tobacco protein in the same systems. RudP carboxylase from barley consists of two different kinds of subunits with the same molecular weight properties as described for other plants. The amino acid composition of the native protein shows similarities with another monocotyledon, oat, both having lower contents of leucine and tyrosine than the dicotyledons, spinach and tobacco. The high content of tryptophan in barley RudP carboxylase gives a higher extinction at 280 nm than has been reported for other organisms (E 1%o = 2.06). This paper also describes a mapping technique for the tryptic peptides of the su-280 nm bunits of RudP carboxylase by two-dimensional high voltage paper electrophoresis which is rapid, reproducible and gives well defined spots. The peptide mapping technique is well suited as a screening method for RudP carboxylase mutants.

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Section: Crystallizationmentioning

confidence: 68%

Section: Smallmentioning

confidence: 99%

Section: Protein Crystallizationmentioning

confidence: 99%

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Ribulose-1,5-diphosphate carboxylase from barley (Hordeum vulgare)

Strøbæk

Gibbons

1976

Carlsberg Res. Commun.

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“…In green plants, the enzyme is found inside the chloroplast, sometimes in crystalline form (1,2). The enzyme can also be crystallized in vitro (3). Our study of crystal form I of the in vitro crystals yielded a preliminary picture of the subunit organization of the molecule.…”

mentioning

confidence: 99%

Ribulose Bisphosphate Carboxylase: A Two-Layered, Square-Shaped Molecule of Symmetry 422

Baker

Eisenberg

Eiserling

1977

Science

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Electron micrographs and x-ray diffraction patterns of crystals of ribu-lose bisphosphate carboxylase, probably the most abundant protein on earth, have provided new details of the arrangement of subunits. The eight large subunits and eight small subunits are clustered in two layers, perpendicular to a fourfold axis of symmetry. Viewed down thefowfold axis, the molecule is square-shaped.Electron micrographs and x-ray diffraction from a new crystal form of oribulose-1 ,5-bisphosphate carboxylase (RuBPCase) show much about the arrangement of subunits in the molecule. RuBPCase initiates the Calvin cycle of photosynthesis and is probably the most abundant protein on earth (1). In green plants, the enzyme is found inside the chloroplast, sometimes in crystalline form (1, 2). The enzyme can also be crystallized in vitro (3). Our study of crystal form I of the in vitro crystals yielded a preliminary picture of the subunit organization of the molecule. The 560,000-dalton enzyme contains eight large (L) (about 55,000 daltons) and eight small (S) (about 15,000 daltons) subunits, and displays one cylindrical hole of about 20 A in diameter that runs through the molecule. The subunits are arranged with a minimum D 2 (222) symmetry; that is, the polypeptide chains are packed around three mutually perpendicular twofold rotation axes. Electron micrographs of a new crystal form, II, show images of the molecules in which some substructure is visible. X-ray diffraction and related measurements on form II show that the eight LS subunit pairs are organized in a two-layer molecule with D4 4 (422) symmetry.Crystal form II of RuBPCase from tobacco is grown by the same procedure as form I (4), except that dialysis of the protein solution (5 to 10 mg/ml) is carried out in 0.05M potassium phosphate buffer at pH 6.0. Two crystal morphologies are obtained: large (0.2 to 0.7 mm), birefringent triangular prisms suitable for x-ray diffraction experiments, and thin (~ 0.1 μm) square platelets suitable for electron microscopy.X-ray precession photographs of the hkO (Fig. 1, A and B) and hOI (not shown) zones of form II reveal that the crystal system is tetragonal with unit cell dimensions a = b = 230 ± 2 Å, and c = 315 ± 3 Å. Optical diffraction patterns (Fig. 1, C and D) from electron micrographs also reveal two perpendicular cell edges of 230 Å. Both x-ray and optical diffraction patterns show that the structure at very low resolution (~ 80 Å) has unit cell dimensions a = b = 162 Å. At higher resolution (15 to 80 Å; Fig. 1 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript determined from the reciprocal lattice symmetry (4/mmm) and systematically absent reflections.In determining the arrangement of molecules within this unit cell we were aided by the micrograph of a negatively stained platelet that is reproduced in Fig. 2. This micrograph is also the first one that we obtained in which some molecular substructure of RuBPCase is visible (see below). The view is down the c-axis, and the dominant feature is an arra...

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“…It was some time after Nobumaro had to resume his duties in Japan that Pak Hoo Chan, Katsuhiro Sakano, and Shalini Singh developed a simple procedure for large scale preparation of crystalline Fraction 1 protein from tobacco leaves (Chan et al 1972; Figure 3 shows a lab photograph of Sakano, Shain-dow Kung, Bentley Atchison and Sam Wildman). The simple procedure provided the means whereby Fraction 1 protein was repeatedly recrystallized without reduction in ribulose-1,5-bisphosphate carboxylase specific activity and proved to possess carboxylation activity as an inherent part of its structure (see Figure 4, reprinted from Wildman and Kwanyuen 1978).…”

Section: The Calvin-benson Studiesmentioning

confidence: 99%

Along the trail from Fraction I protein to Rubisco (ribulose bisphosphate carboxylase-oxygenase)

Wildman

Discoveries in Photosynthesis

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This historical minireview deals with events leading to the eventual discovery of Rubisco (ribulose bisphosphate carboxylase-oxygenase). This abundant leaf protein is not only responsible for the net fixation of CO 2 in all plants, but also causes the loss of carbon through photorespiration. The latter is a special 'problem' of the so-called 'C 3 ' plants. The protein was first called 'Fraction 1 protein' before it was recognized to be the same as Rubisco. Instead of reinventing words, text as needed has been freely used from three earlier publications (Wildman and Kwanyuen

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Crystalline Fraction I Protein: Preparation in Large Yield

Cited by 132 publications

References 3 publications

Ribulose-1,5-diphosphate carboxylase from barley (Hordeum vulgare)

Ribulose-1,5-diphosphate carboxylase from barley (Hordeum vulgare)

Ribulose Bisphosphate Carboxylase: A Two-Layered, Square-Shaped Molecule of Symmetry 422

Along the trail from Fraction I protein to Rubisco (ribulose bisphosphate carboxylase-oxygenase)

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