Audree V. Fowler

Amino acid sequence of a probable amylase/protease inhibitor from rice seeds.

The primary structure of a 9-kDa basic protein from rice seeds was determined by gas-phase sequencing of the intact protein and peptides derived from it by digestion with trypsin, chymotrypsin, and endopeptidase Lys-K. The protein consists of a single polypeptide chain of 91 amino acid residues with a calculated molecular mass of 8909 Da. It is rich in alanine, serine, glycine, and cysteine. The eight cysteines form four disulfide bonds. There is no methionine, histidine, phenylalanine, or tryptophan. The sequence is highly homologous with an alpha-amylase inhibitor, I-2, from seeds of Indian finger millet [F. A. P. Campos and M. Richardson (1984) FEBS Lett. 167, 221-225] and a 10-kDa barley seed protein, also called a probable amylase/protease inhibitor [B. Svensson et al. (1986) Carlsberg Res. Commun. 51, 493-500; J. Mundy and J. C. Rogers (1986) Planta 169, 51-63]. In analogy with the barley protein, the purified protein is tentatively called a rice probable amylase/protease inhibitor (PAPI). The rice PAPI does not show inhibitory activities against proteases and amylases tested. The amino acid sequence is as follows: Ile-Thr-Cys-Gly-Gln-Val-Asn-Ser-Ala-Val(10)-Gly-Pro-Cys-Leu-Thr-Tyr- Ala-Arg-Gly-Gly(20)-Ala-Gly-Pro-Ser-Ala-Ala-Cys-Cys-Ser-Gly(30)-Val-Arg- Ser-Leu-Lys-Ala-Ala-Ala-Ser-Thr(40)-Thr-Ala-Asp-Arg-Arg-Thr-Ala-Cys- Asn-Cys(50)-Leu-Lys-Asn-Ala-Ala-Arg-Gly-Ile-Lys-Gly(60)-Leu-Asn-Ala-Gly- Asn-Ala-Ala-Ser-Ile-Pro(70)-Ser-Lys-Cys-Gly-Val-Ser-Val-Pro-Tyr-Thr(80)- Ile-Ser-Ala-Ser-Ile-Asp-Cys-Ser-Arg-Val-Ser(91).

Co-linearity of β-Galactosidase with Its Gene by Immunological Detection of Incomplete Polypeptide Chains

Many nonsense mutants that map in the β-galactosidase structural gene produce material that forms precipitin lines when tested by double diffusion on agar against antiserum prepared from native β-galactosidase. Relative sizes of the cross-reacting materia!s measured by sucrose density gradient centrifugation are the same as sizes calculated from genetic mapping of nonsense mutants. Orientation of the protein to its gene is also indicated.

Specificity of endoproteinase Asp-N (Pseudomonas fragi): Cleavage at glutamyl residues in two proteins

Endoproteinase Asp-N, a metalloprotease from a mutant strain of Pseudomonas fragi, has been reported to specifically cleave on the N-terminal side of aspartyl and cysteic acid residues. We utilized this enzyme to generate fragments for determining the amino acid sequence of the D-aspartyl/L-isoaspartyl methyltransferase isozyme I from human erythrocytes. Surprisingly, we identified cleavage sites for this enzyme at the N-terminal side of several glutamyl residues in addition to the expected cleavage sites at aspartyl residues. The ability of this enzyme to cleave polypeptides at both glutamyl and aspartyl residues was confirmed by mapping additional sites on erythrocyte carbonic anhydrase I. These results indicate that a more appropriate name for this enzyme may be Endoproteinase Asp/Glu-N.

β-Galactosidase: Immunological studies of nonsense, missense and deletion mutants

Abstract A series of nonsense mutants which map throughout the β-galactosidase structural gene, and several missense and deletion mutants, were analysed for antigenic activity with purified antiserum produced from native β-galactosidase. Quantitative analyses by the Ouchterlony test, by inhibition studies, and by microcomplement fixation are in general agreement. These show, relative to the position of the nonsense mutation, no immunological activity from mutants of the early (operator) part of the gene, a peak at the center, next a much lower level, and finally a relatively high level in mutants mapping in the terminal part of the gene. No antigenic activity was found in a deletion mutant which produces both the alpha and omega fragments of β-galactosidase. Examination of precipitin lines on Ouchterlony plates of nonsense, missense and deletion mutants, as well as other data, suggests the presence of three classes of antigenic sites, those of relatively long chains, those of relatively short polypeptide chains and, tentatively, those due to the polymer. Heat treatment or addition of sodium dodecyl sulfate destroys immunological activity of nonsense mutant protein. It is suggested that variations in level of antigenic activity of incomplete β-galactosidase chains are due not only to the presence or absence of antigenic sites, but in addition to differences in conformation of these chains.

β -Galactosidase, the Lactose Permease Protein, and Thiogalactoside Transacetylase

INTRODUCTION The three structural genes of the lac operon, Z , Y , and A , code for β -galactosidase, the lactose permease protein, and thiogalactoside transacetylase, respectively. Since the discussion of these gene products in The Lactose Operon (Zabin and Fowler 1970; Kennedy 1970), a considerable amount of work has been carried out, particularly on β -galactosidase. In 1970 the subunit structure of this enzyme was known with reasonable certainty, but some controversy remained. Now the primary structure has been completed. Much new information is available on β -galactosidase from strains with mutations in the lacZ gene, on complementation, and on immunological properties. Interesting fusion proteins have been described. Considerably less information has been accumulated on the lacY gene product, and in fact, the lactose permease protein has not yet been obtained in chemically pure form. The function of thiogalactoside transacetylase has been a mystery for a long time. Recently evidence has been presented for a possible physiological role in the cell. A detailed study of its enzymatic characteristics has been carried out, and a beginning has been made toward determining the amino acid sequence of this protein. Such information on the three proteins is presented here and is discussed primarily from a biochemical point of view. β -GALACTOSIDASE Production and Isolation β -Galactosidase ( β -D-galactoside galactohydrolase E. C. 3.2.1.23) accounts for up to 5% of the total protein in haploid strains of Escherichia coli. Considerably higher levels can be obtained from certain partial diploids (Fowler 1972a). When the strain A324–5 was grown on minimal medium containing...

The cerebroside sulfate activator from pig kidney: Purification and molecular structure

The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.

Gene fusion techniques cloning vectors for manipulating lacZ gene fusions

Abstract A simple vector system for cloning gene fusions of lacZ is described. We apply one of these new vectors to the cloning and transcriptional analysis of the promoter region of the ompF gene of Escherichia coli .

Production of α-hydroxy fatty acids by two strains of Lactobacillus casei☆

Abstract The principal α-hydroxy acids other than lactic acid produced by L. casei strains 7469 and 280-16A were isolated by countercurrent extraction and identified as α-hydroxyisocaproic acid and α-hydroxyisovaleric acid. The amount of these acids and of lactic acid were estimated from counter-current distribution data, and the degrees of racemization were estimated from specific rotation values and from the results of optically specific microbiological assays. The maximal concentrations of α-hydroxyisocaproic and α-hydroxyisovaleric acids produced by L. casei were approximately 0.6 and 0.2 μeq./ml., respectively. The lactic acid, α-hydroxyisovaleric acid, and α-hydroxyisocaproic acid produced by L. casei 7469 were approximately 16%, 62%, and 97% racemic (the rest l -), respectively, whereas the same acids produced by L. casei 280-16A were much less racemic. This was particularly true of the L. casei 280-16A lactic acid, which appeared to be 99.3% l -lactic acid. Racemization of the α-hydroxy acids appears to be of importance in L. casei since strain 280-16A, which produces only slightly racemized acids, fails to grow in the usual media unless provided with a d -α-hydroxy acid source.

The amino acid sequence of thiogalactoside transacetylase of Escherichia coli

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.

Purification of thiogalactoside transacetylase by affinity chromatography.

Thiogalactoside transacetylase, the product of the lacA gene of the lactose operon of Escherichia coli, has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration.