Abstract
Acidic water stress polypeptides (Wsp) with molecular masses of 33, 37 and 39 kDa are the most abundant soluble proteins in the cyanobacterium Nostoc commune. Wsp polypeptides and UV-A/B-absorbing pigments are secreted by cells, accumulate in the extracellular glycan sheath, and are released from desiccated colonies upon rehydration. No evidence was obtained for either glycosylation, phosphorylation, or acylation of Wsp polypeptides. NH2-terminal amino acid sequences of the 33-, 37-, and 39-kDa polypeptides were identical: Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu-Gln-X-Ile-Gln-Asn-Pro-Ser-Asn- Pro-Ser-Asn-Gly-Lys-Gln. This consensus NH2-terminal sequence and an internal sequence (Glu-Ala-Arg-Val-Thr-Gly-Pro-Thr-Thr-Pro-Ile-Asp) showed homologies with the sequences of carbohydrate-modifying enzymes. Purified Wsp polypeptides associate with a 1,4-beta-D-xylanxylanohydrolase activity that was inhibited specifically by Wsp antiserum. In the absence of salt, Wsp polypeptides, and the water-soluble UV-A/B-absorbing pigments, form multimeric complexes through strong ionic interactions. A possible role is suggested for Wsp polypeptides in the synthesis and/or modification of a xylose-containing UV-A/B-absorbing pigment.
Highlights
From the $Department of Biochemistry a n d Anaerobic Microbiology, Virginia Polytechnic Institute and StateUniuersity, Blacksburg, Virginia 24061 a n d the gznstitut fur Mikrobiologie, Technische Universitat Munchen, 0-85350Freising, Germany
W-AB-absorbingpigments, form multimeric complexes weights of desiccated colonies were suspended in sterile water with or through strongionic interactions.A possible roleis suggestedfor waterstress polypeptides (Wsp) polypeptides in the synthesis andor modification of a xylose-containing W-"absorbing pigment
Peptide Mapping and Sequence Analysis-Individual Wsp polypeptideswere excised from SDS-PAGE gels and electroeluted.Protein samples were dialyzed exhaustively against water using 3500 cut-off dialysis membrane
Summary
Tergent MEGA 8 (Sigma) was included to prevent denaturationof the endoglycosidase by SDS [13].Aliquots of the supernatantfraction were diluted 1:20 with a n endoglycosidase digestion buffer(80mM Tris-HC1, pH 6.8, 1.7%(vlv) P-mercaptoethanol, 1%(wlv) MEGA 8) prior to the addition of 5 units of peptide-N-glycosidase F Wsp I s Secreted Beyondthe Outer Cell Membrane-Colonies of N . Peptide Mapping and Sequence Analysis-Individual Wsp polypeptideswere excised from SDS-PAGE gels and electroeluted.Protein samples were dialyzed exhaustively against water using 3500 cut-off dialysis membrane In all cases where Wsp was detected by Westernblotting, it wasjudged to be the most abundant soluble protein following purification of cell extracts (Fig. 2, laneI; see alsoFig. 2a, Ref. 11).For three samplesfrom. Enzyme Assays-The capacity of native extractsof Wsp to hydrolyze peptides dration, were strongest following approximately irrespective of the presence or absence. BecauseWsp polypeptides may stillhave been present in the sheath despitemultiple rounds of aqueousextraction(see Fig. 1 E ) it is difficult to assay (Pierce Chemical Co. publication 23200) was used to estimate assess with certainty the intracellucloanrcentration of Wsp
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