Associated with some plant viruses are small satellite RNA's that depend on the plant virus to provide protective coat protein and presumably at least some of the proteins necessary for satellite RNA replication. Multimeric forms of the satellite RNA of tobacco ringspot virus are probable in vivo precursors of the monomeric satellite RNA. Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA. The reaction generates biologically active monomeric satellite RNA, apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation.

The glycosidase activities associated with Xenopus laevis eggs were determined using p‐nitrophenyl glycosides as substrates. The egg lysate contained significant amounts of α‐fucosidase, N‐acetyl‐β‐D‐galactosaminidase, and α‐mannosidase activities with smaller amounts of other glycosidase activities, including N‐acetyl‐β‐D‐glucosaminidase. A cortical granule exudate obtained from ionophore‐activated dejellied eggs contained predominantly glucosaminidase activity with only trace amounts of other glycosidase activities. Perivitelline space material obtained from activated or fertilized jellied eggs contained only glucosaminidase activity. Using cytochemical staining procedures, glucosaminidase activity was present in the perivitelline space and the inner aspect of the jelly coat after fertilization or activation of the egg, but not before. The rate of glucosaminidase activity released from activated eggs occurred with the same kinetics as the cortical reaction. The cortical granule and noncortical granule glucosaminidase activities had different electrophoretic mobilities as determined by disc gel electrophoresis. Thus, the Xenopus laevis egg has two N‐acetyl‐β‐D‐glucosaminidase activities, one associated with the cortical granules and the other associated with the noncortical granule compartment of the cell. The cortical granule enzyme released from the egg at fertilization may function in altering the egg's penetrability to supernumerary sperm.

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