Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl‐blocked protein structures

Egestad, Börje; Estonius, Mats; Danielsson, Olle; Persson, Bengt; Cederlund, Ella; Kaiser, Rudolf; Holmquist, Barton; Vallée, Bert L.; Parés, Xavier; Jefferey, Jonathan; Jörnvall, Hans

doi:10.1016/0014-5793(90)81152-e

Cited by 12 publications

(17 citation statements)

References 8 publications

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“…Interestingly, N-terminal acetylated or acylated GGPDH sequences have already been described for various other organisms (Egestad et a/., 1990;Jeffery et a/., 1989).…”

Section: Purification and Characterization Of Potato G6pbhmentioning

confidence: 91%

Purification, characterization, and cDNA sequence of glucose‐6‐phosphate dehydrogenase from potato (Solatium tuberosum L.)

1994

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Summary Glucose‐6‐phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) has been purified from potato tuber at least 850‐fold to apparent homogeneity as judged by SDS‐PAGE. The enzyme was characterized by Km values of 260 μM for glucose‐6‐phosphate and 6 μM for NADP and a broad pH optimum between phi 7.5 and 9. NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity. Dithiothreitol (DTT) did not inactivate the enzyme. A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots. A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh‐specific primers. A full‐length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources. The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%). The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox‐independent cytosolic isoform was obtained.

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“…Interestingly, N-terminal acetylated or acylated GGPDH sequences have already been described for various other organisms (Egestad et a/., 1990;Jeffery et a/., 1989).…”

Section: Purification and Characterization Of Potato G6pbhmentioning

confidence: 91%

Purification, characterization, and cDNA sequence of glucose‐6‐phosphate dehydrogenase from potato (Solatium tuberosum L.)

1994

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“…All positions have been positively identified, including the N-terminal blocking acetyl group [15]. Notably, it was possible also to deblock the whole protein chain using trifluoroacetic acidmethanol and subsequently to identify the N-terminal region after Edman degradation.…”

Section: Discussionmentioning

confidence: 99%

“…Peptide T1 did not undergo Edman degradation, indicating that its N-terminus is blocked, and fast atom bombardment MS of this peptide had already shown it to be N-terminally acetylated [15]. Deblocking was achieved by treat- ment of the intact protein with trifluoroacetic acidmethanol (1 : 1) for 16 h in an evacuated tube at room temperature.…”

Section: N-terminusmentioning

confidence: 99%

Glucose‐6‐phosphate dehydrogenase

Jeffery

Persson

Wood

et al. 1993

European Journal of Biochemistry

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The primary structure of glucose-6-phosphate dehydrogenase from the yeast Pichia jadinii (formerly Candidu utilis) has been determined. It consists of a 495-residue, N-terminally acetylated protein chain. The structure shows extensive differences from those of the corresponding mammalian, fruit fly, and bacterial enzymes (52-68% residue non-identities), but also from that of another yeast, Saccharornyces cerevisiae (38 %).A eubacterial type and a yeast type of glucose-6-phosphate dehydrogenase are discerned, in addition to the known mammalian type. They are distinguished from each other, from the mammalian type, and the insect enzyme, on the basis of both specific residues and pattern differences.The distribution of residues conserved in all forms locates short segments in which identities are closely grouped. Approximately 50% of these segments correspond to predicted turns and appear to mark the principal folds characteristic of the enzyme's tertiary structure. A region in the Nterminal part of the protein chain has characteristics suggestive of a coenzyme-binding site, while, in the middle third, another functionally important segment may be related to glucose-6-phosphate binding and catalysis.Glucose-6-phosphate dehydrogenase directs glucosedphosphate into the pentose-phosphate pathway and plays a pivotal role in cell function. Structural similarities between glucose-6-phosphate dehydrogenases and other proteins analyzed are slight [ l], and the characterized glucose-6-phosphate dehydrogenases differ within a frame of clear homology. The mammalian (both eutherian and metatherian) forms of the enzyme differ only marginally (around 10% variation of primary structures among human [2], rat [3], and opossum [4] glucose-6-phosphate dehydrogenases), while more discriminating differences (38 -52 %) are observed when the set is extended to include the fruit fly [5] and Bakers' yeast [6] enzymes. We have now determined the structure of Pichia jadinii glucose-6-phosphate dehydrogenase, which allows examination of the enzymes from genetically well separated yeasts (P. jadinii and Saccharornyces cerevisiae), permits further development of functional interpretations, and makes it possible to distinguish structurally distinct subgroups among the enzyme forms known.

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“…1. The acetyl-blocking group was identified by mass spectrometry using fast atom bombardment, showing the molecular mass of K1 to be 533.3 Da and that of El to be 1832.3 Da [29].…”

Section: Structural Analysismentioning

confidence: 99%

“…However, extractive losses of the highly hydrophobic C-terminal segment prevented good recovery of the thiohydantoin derivative from the C-terminal leucine residue. Nevertheless, this residue was ascertained in the total composition of both peptides (Supplement Table 2 for E16), and by the molecular mass of El6 determined to be 1875.55 Da by fast-atom-bombardment mass spectrometry [29], thereby establishing the Cterminal assignment. Furthermore, the presence of the Cterminal Leu was confirmed by the amino acid sequence deduced from the cDNA sequence (below).…”

Section: Structural Analysismentioning

confidence: 99%

Avian alcohol dehydrogenase: the chicken liver enzyme

Estonius

Karlsson

Fox

et al. 1990

European Journal of Biochemistry

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The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to y chains of the human enzyme, a K, for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 pM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order I < I11 < 11, and human/chicken differences are less than inter-class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present-day classes I and 11 deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class I11 forms. The coenzymebinding and substrate-binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit-interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and I11 mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.Chicken liver alcohol dehydrogenase was purified early by means of AMP-Sepharose affinity chromatography [l]. Data then obtained suggested that it was related to the mammalian enzyme [2], which was at that time still not recognized as subdivided into three classes [3]. Class I represents the traditional, pyrazole-sensitive liver enzyme (reviewed in [4]) ; class I1 has a higher K, for ethanol and is less sensitive to pyrazole, while class 111 has little activity towards ethanol and is almost insensitive to pyrazole. The classes constitute distinct enzyme types whose distributions and structures are characteristic [3, 5, 61. The evolutionary properties defined the class 111 form as an enzyme separate from class I forms [6], and it has recently been shown also to exhibit glutathione-dependent formaldehyde dehydrogenase activity [7]. The large differences between the mammalian alcohol dehydrogenase classes and the lack of specific substrates for the class I and I1 forms make studies of sub-mammalian vertebrate forms important to trace both their evolutionary relationships and possibly their original functional roles.

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Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl‐blocked protein structures

Cited by 12 publications

References 8 publications

Purification, characterization, and cDNA sequence of glucose‐6‐phosphate dehydrogenase from potato (Solatium tuberosum L.)

Purification, characterization, and cDNA sequence of glucose‐6‐phosphate dehydrogenase from potato (Solatium tuberosum L.)

Glucose‐6‐phosphate dehydrogenase

Avian alcohol dehydrogenase: the chicken liver enzyme

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