The preparation and purification of cytidine 2′:3′-phosphate

Crook, E. M.; Mathias, A. P.; Rabin, B. R.

doi:10.1042/bj0740230

Cited by 48 publications

(38 citation statements)

References 6 publications

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“…Initial velocity measurements of the hydrolysis of Cyd-2': 3'-P catalyzed by RNase A and its fluorescein thiocarbamyl derivative were performed spectrophotometrically at 292 nm, using an absorbance difference of 570 M-' cm-' between Cyd-2': 3'-P and Cyd-3'-P at this wavelength [9].…”

Section: Activity Measurementsmentioning

confidence: 99%

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Garel

1976

Eur J Biochem

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A chemical derivative of bovine pancreatic ribonuclease A (RNase A) has been prepared by reaction with fluorescein-isothiocyanate at pH 6. This derivative has a fluorescein group covalently attached to the a-amino group of the protein. The enzymic properties of the modified protein are similar to those of RNase A. It is shown that the pK of the fluorescein group can be used as an index of protein conformation to monitor structural changes in the protein. In this work, the binding of a specific inhibitor (cytidine 2'-monophosphate) to RNase A, the isomerization process occurring in RNase A around pH 6, and the thermal unfolding of RNase A, were studied by mean of the pK changes of the fluorescein group. The results obtained by this method are fully consistent with those obtained by other methods. It is proposed that using ionizable reporter groups and their changes in pK to monitor conformational changes in proteins may be a sensitive tool both in equilibrium and kinetic studies.

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Section: Activity Measurementsmentioning

confidence: 99%

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Garel

1976

Eur J Biochem

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“…The measurement of hydrolysis of cCMP (cCMP assay) was carried out similarly to Crook et al [12]. Samples of 10-100 pl were added to 2 ml of 0.1 or 0.2 mg/ml cCMP in 10 mM Tris (pH 7.9, 0.15 M KCl, 2 mM MgC12 and the decrease of absorbance at 284 nm of the mixture was recorded.…”

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confidence: 99%

On the interaction of bovine seminal RNase with actin in vitro

Simm¹,

Krietsch²,

Isenberg³

1987

Eur J Biochem

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Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it (1) binds to actin with an apparent binding constant of 9.2 x lo4 M-' in 0.1 M KC1, (2) induces the polymerization of actin below the critical concentration in depolymerization buffer, (3) accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than l / l O O , and (4) bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. (5) Actin inhibits the enzymatic activity of RNase BS.RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin.We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.Many proteins have been discovered which interact with actin, the major component of the eucaryotic cytoskeleton [l]. Most of these proteins were isolated with the aim of investigating their regulating effect on actin polymerization in vitro and the possible interconversion of different actin structures inside the cell.Among those the protein pancreatic DNase I gained special attention, since it was found that DNase I depolymerizes F-actin to form a 1 : l complex with G-actin [2], although its physiological relevance remains questionable. In this context it was of interest to look for a possible interaction of the complementary protein pancreatic RNase A with actin. We show here that only the dimeric RNase [3,4], a cytotoxic substance [5,6] from bovine seminal plasma, strongly interacts with actin as a gelation, bundling, and nucleation factor. MATERIALS AND METHODS Preparation of proteinsAll preparations were carried out at 4°C. Rabbit skeletal muscle actin was prepared according to Spudich and Watt [7].

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“…Specific activities were determined by the methods of Crook et al [6] [8]. Cyclic cytidylate was dissolved in 0.1 M TrissHCl/O.l M NaCl, pH 7.0 (1.02 X 1 0e4 M).…”

Section: Assay Of Rnase a And Rnase Pmentioning

confidence: 99%