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An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.

A method for the removal of pyridoxal 5'-phosphate (PLP) from rabbit muscle phosphorylase b is described. The procedure involves two simultaneous operations, namely, distortion of the protein by an appropriate deforming agent which exposes the covalently bound PLP, and removal of the cofactor by interaction with a PLP reagent. A number of deforming buffers were tested, of which imidazolium citrate proved to be the most effective. Using this buffer and L-cysteine as the carbonyl reagent, resolution of phosphorylase occurred with a half-life of ca. 5 min at pH 6.2, O", <1 min at pH 7.0 and 37"; the energy of activation at pH 7.0 was 11.7 kcaljmole. Resolution was specific with respect to the PLP reagent. Of a number of cysteine analogs tested, L-cysteine was found to be the most effective; penicillamine was barely active and cysteamine totally inactive. Under these same condi-I n the first paper of this series (Hedrick and Fischer, 1965) phosphorylase (EC 2.4.1.1 a-1,4-glucan: orthophosphate glucosyltransferase) was shown to display none of the enzymatic activities characteristic of B6-dependent enzymes (for a recent review, see Meister, 1965). It was also reported that in nonenzymatic model systems, PLPl and several of its analogs or derivatives had no catalytic effect on the reaction between glucose 1-phosphate and glycogen. Removal of PLP from phosphorylase was shown to yield an enzymatically inactive protein . On the other hand, reduction of phosphorylase b with sodium borohydride, resulting in a fixation of the cofactor onto

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The extracellular matrix (ECM) surrounding the anuran egg is composed of jelly coat layers, an envelope, and the perivitelline space, which separates the envelope from the egg plasma membrane. Both the jelly coat layers and egg envelopes are required for fertilization in anurans. This paper reviews the current understanding of the structure-function relations of the ECM, with emphasis on the egg envelope. The fibrous egg envelope exists in four related forms. The envelope forms differ in their ultrastructures, macromolecular compositions, and cellular functions. After the oocyte is released from the ovary, conversion of one envelope form to another is brought about by factors secreted by the oviduct prior to fertilization and by factors released from the egg in the sperm-triggered cortical reaction. An additional extracellular matrix structure, located in the perivitelline space, has recently been identified in Xenopus laevis, as well as a previously undescribed reorganization of envelope fibers occurring at fertilization. The molecular changes in the ECM glycoproteins (limited proteolysis, lectin-ligand binding, and conformational changes) and the oviductal and egg macromolecules responsible for the conversion of envelope forms are discussed. New experimental evidence that supports the lectin-ligand hypothesis for the formation of the fertilization layer is presented. It is proposed that the molecular changes in the ECM are responsible for the ultrastructural alterations of the ECM and for modifications of the fertilization and developmental functions of the anuran egg ECM.