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Abstract
Abstract Evidence is presented documenting the nuclear location of the enzyme, cytidine 5'-monophosphosialic acid (CMP-sialic acid) synthetase. This enzyme catalyzes the reaction: [see PDF for equation] From previous studies it had been inferred that this enzyme was cytoplasmic. Studies are reported here that describe the following. (a) The purity of the nuclei that were used in these investigations. Nuclei were isolated from hypertonic sucrose solutions from the retina of the hog and from liver, kidney, spleen, and brain of the rat. Nuclear purity (i.e. absence of cytoplasmic contamination) was established on the basis of enzymatic, chemical, and morphological criteria. (b) The activity of CMP-sialic acid synthetase in these nuclei. The yields of CMP-sialic acid synthetase in the nuclei recovered from the different tissues varied from 18 to 53% of the activities present in the initial homogenates. When corrected for the yield of nuclei, based on DNA content, the yields of enzyme ranged from 54 to 90%. This was accompanied by increases in specific activities of 3- to 13-fold. CMP-sialic acid synthesized by nuclei from the retina of the hog was identified by chemical, enzymatic, chromatographic, and electrophoretic procedures. (c) Some of the properties of nuclear CMP-sialic acid synthetase are described. The following apparent Km values were observed for the enzyme from nuclei of rat liver: N-acetylneuraminic acid, 0.72 mm; N-glycolylneuraminic acid, 1.4 mm; CTP, 0.48 mm; Mg2+, 6.75 mm. The optimal pH for the reaction is between 8.3 and 8.6 in Tris buffer. The effect of other metals and nucleotide triphosphates on the activity of the enzyme was measured.
Highlights
Since some RNA is indigenous to the nucleus, its presence cannot be regarded exclusively as a sign of cytoplasmic contamination [33]
As a result of analyses of this type, the nuclei from both hog retina and rat liver used in the present studies can be described as being relatively “pure”; i.e. they contained little, if any, cytoplasmic contamination
Least one form of the sialic acid-activating enzyme exists as a nuclear enzyme and in relatively high concentration
Summary
Grant EY 00393 from the National Eye Institute, United StatesPublic Health Service, the Ohio Lions Eye Research Foundation, and Public Health Service General Research Support GrantFR-5410 from the National Institutes of Health.An abstract of some of these studies has been published [1].Research, Case Western Reserve University, Cleveland, OhioN-GN, N-glycolylneuraminic acid; N-AN-aldolase, the specific aldolase that cleaves N-AN and N-GN to pyruvate and N-acylmannosamine; TBA, thiobarbituric acid.N-Acetylneuraminic acid and nucleotide triphosphates were obtained from Calbiochem. Grant EY 00393 from the National Eye Institute, United States. Public Health Service, the Ohio Lions Eye Research Foundation, and Public Health Service General Research Support Grant. FR-5410 from the National Institutes of Health. An abstract of some of these studies has been published [1]. N-GN, N-glycolylneuraminic acid; N-AN-aldolase, the specific aldolase that cleaves N-AN and N-GN to pyruvate and N-acylmannosamine; TBA, thiobarbituric acid. N-Acetylneuraminic acid and nucleotide triphosphates were obtained from Calbiochem. DNA (calf thymus) and phenazine methosulfate were obtained from General Biochemicals. N-Glycolylneuraminic acid was prepared by the procedure of Brunetti, Swanson, and Roseman [12]. The author is grateful to Dr Saul Roseman for a sample of N-AN-aldolase purified from Clostrid~um perfringens[12]
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