Abstract
Saccharomyces cerevisiae external and internal invertases have been amplified by introducing the normal and modified SUC2 genes into yeast multicopy plasmids, which were then used to transform a yeast strain resistant to repression by glucose. Amino acid compositional analysis of these enzymes, in addition to end group sequencing, confirmed the DNA sequence data of Taussig and Carlson (Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954), indicating that both enzymes were encoded in the same gene. Comparison of the properties of carbohydrate-containing external invertase and its nonglycosylated internal form revealed that although the carbohydrate did not appear to influence the conformation of the peptide backbone, as determined by circular dichroism analyses, its presence considerably enhanced the ability of guanidine HCl-denatured external invertase to be renatured relative to internal invertase. The Mr of the internal enzymes was found to be greatly dependent on pH with the enzyme being a monomer at pH 9.4, a dimer at pH 8.3, and an apparent octamer at pH 4.9.
Highlights
Plasmids, which were used to transform a yeast strain resistant to repression by glucose
By employing recombinant DNA techniques whose synthesisis repressed by glucose, is located in the we have overcome the problem of quantity by amplifyingboth periplasmic space and contains 50% of its mass as polyman- the internal anedxternal productsof the SUC2 gene to a level nan, while the other, which is devoid of carbohydrate, is representing about1%of the yeast cellular protein
The extremely low level of internal invertase and the large amount of carbohydrate associated with external invertase contributed to the problem of understanding the origin of these available, we have been able to compare their properties and to clearly confirm by end group and amino acid analysis that thetwo enzymes are derived from the same gene
Summary
Denaturation of internal and external (native or partially deglycosylated) invertase samples (100-200 pg/ml) was examined by measuring enzyme activity following incubation with increasing concentrations (0-3 M) of guanidine HCl a t 50 mM sodium acetate buffer, pH 5.0, for 30 min a t 4 “C. Invertase activity was assayed in the presence of the respective concentrations of guanidine HCl as described below. Denaturation of internal and external (native or partially deglycosylated) invertase samples (100-200 pg/ml) was examined by measuring enzyme activity following incubation with increasing concentrations (0-3 M) of guanidine HCl a t 50 mM sodium acetate buffer, pH 5.0, for 30 min a t 4 “C. The reversibility of denaturation was studied following preincubation of enzyme with 2.5 M guanidine HC1a t 4 “C.At various times (0-2 h) aliquots were diluted 500-fold with ice-cold buffer and assayed for invertase activity. To amplify internal invertase synthesis,the SUC2 gene was modifiedso that an mRNA encoding internal invertase was transcribed from the external invertase promoter This was achieved by deleting a 37-bp HindIII fragment, which contains the start codon for the signal peptide (Fig. 1).Yeast cells transformed with the plasmid pRT9 containing the modified SUC2 gene produce high levels of nonglycosylated invertase (Fig. 2). The difference in mobility between the FH4C and S288C internal invertase noted in the present study (Fig. 2, lane 1 versus lune 2 ) will be discussed in subsequent sections
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