Sigmund R. Suskind

The similar properties of tryptophan synthetase and a mutationally altered enzyme in Neurospora crassa

A method has been developed for the stabilization and further purification of tryptophan synthetase from N. crassa. The sedimentation constant, diffusion coefficient and approximate molecular weight of the enzyme from strain C-84 have been determined. A marked similarity in physical properties was found between the enzyme and an antigenically related protein in one tryptophan-requiring mutant. These properties include behavior during purification, elution pattern during DEAE cellulose column chromatography, and sedimentation velocity constants. The results support the hypothesis that the antigenically related proteins in the td mutants are in fact genetically altered tryptophan synthetase molecules.

Ribonucleic acid synthesis and thymineless death.

Abstract Conditions which uncouple the net synthesis of RNA and of protein in the absence of thymine have been examined in E. coli B3 to determine whether either of these macromolecular syntheses is specifically correlated with thymineless death. Thymineless death can occur when protein synthesis and cell growth are strongly inhibited. Under such conditions the extent of thymineless death is closely correlated with the unbalanced synthesis of RNA. Death can be prevented by inhibiting RNA synthesis. The nucleotide composition of RNA synthesized during thymineless death does not differ significantly from the RNA made during normal growth in the presence of thymine.

ANTIENZYMES IN IMMUNOGENETIC STUDIES

A. Anti-enzymes, those antibodies formed against enzymatically active proteins, offer an exceedingly useful probe for studying certain aspects of the genetic control of enzyme formation, structure and function. In addition to the important attributes of antigen-antibody reactions in general, i.e., their specificity and sensitivity, the ability of anti-enzymes to precipitate and often inhibit enzymic activity provides us with a means of detecting and characterizing individual enzymes in multicomponent systems. While this paper will concentrate primarily on the use of anti-enzyme technics in one microbial system, the wide application of such methods to diverse problems should be noted. These include aspects of enzyme formation, characterization of genetically altered enzymes, alteration of protein structure following the incorporation of amino acid analogs, the specificity of protein structure, studies on regulatory or control processes, cellular fine structure and the localization of enzymes, and isozymes and chemical embryology.l-19 The experimental technics well known to the immunochemist have been successfully applied to the fields of enzymology, protein chemistry, physiology, embryology and genetics. B. It has been established in several cases that primary genetic control is exerted by specification of the amino acid sequence of protein. Several species of RNA have been described which act as intermediates between the original genetic information cai-ried in the DNA, and the phenotypic expression of this information in the form of the completed protein molecule. Where mutations have occurred within a genetic region known to control the formation of a specific protein, the concomitant formation of genetically altered proteins frequently occurs.2o Usually thesc mutant proteins have properties distinct from the normal molecule. Why is the study of mutationally altered proteins of interest to the genetically oriented biochemist or immunochemist and how have antienzymes in particular, proved valuable in chemical genetic research? Antienzymes have been used to detect mutationally altered enzymes which are devoid of, or possess aberrant, enzymic activity. They have provided an excellent means of characterizing the antigenic and immunogenic properties of these abnormal proteins and relating these to other properties of the molecule. In se\reral instances the characteristic properties of the mutant proteins have been related to mutational events occurring within

Enzymatic activity of a genetically altered tryptophan synthetase in Neurospora crassa.

Partially purified preparations of certain tryptophan-requiring mutants of Neurospora crassa, which contain a protein (CRM) antigenically related to wild-type tryptophan synthetase, possess indole glycerol phosphate-synthesizing activity. This activity can be inhibited by anti-CRM sera. It is suggested that CRM in such mutants represents a damaged tryptophan synthetase lacking the capacity to react with l-serine.

Enzymic properties of a mutant tryptophan synthase from Neurospora crassa.

Abstract Tryptophan synthase ( l -serine hydro-lyase (adding indole), EC 4.2.1.20) obtained from Neurospora crassa strain td 201 , a tryptophan auxotroph, is only capable of catalyzing the conversion of indole and serine to tryptophan. The enzyme is largely resolved from its cofactor, pyridoxal phosphate (pyridoxal- P ); hence the enzymic activity in the crude extract is practically undetectable unless exogeneous pyridoxal- P is added. The stoichiometry of the cofactor has been determined by spectrophotometric titration with purified apoenzyme; about 1.5 moles of pyridoxal- P are bound to 1 mole of enzyme. Two types of binding of pyridoxal- P are present in the enzyme; one absorbs at 325 nm ( λ max emission = 385 nm) and the other absorbs at 410 nm ( λ max emission = 495 nm). These two forms are not interconvertible by varying the pH. The holoenzyme can be easily resolved by passage through a hydroxylapatite column. The resulting apoenzyme is inactive but it can be reactivated by pyridoxal- P . NaBH 4 reduction leads to irreversible inactivation of the enzyme. The enzyme is very sensitive to some endogenous proteases present in the Neurospora extract. Phenylmethanesulfonyl fluoride can prevent to some extent the spontaneous inactivation of the enzyme. Highly purified enzyme has been found to be fairly stable. Therefore, the apparent lability of this enzyme is not an intrinsic property. The catalytic activity of purified enzyme can be stimulated by proteins such as serum albumin. This phenomenon has been interpreted in terms of the “intermolecular cooperativity” which might exist in vivo between this enzyme and the other enzymic components of the tryptophan pathway. In this paper, an improved assay method for enzymic activity is also described.

Tryptophan Synthetase from Neurospora: A Modification in the Reaction Scheme

In addition to indole-3-glycerolphosphate, an unreported reaction product has been detected in incubation mixtures containing tryptophan synthetase from Neurospora, indole, and glyceraldehyde-3-phosphate. This product has been tentatively identified as an indole derivative. Since this compound is not formed in incubation mixtures initially containing only enzyme and indole-3-glycerolphosphate, it was concluded that indole-3-glycerolphosphate synthesis and breakdown are not simply the forward and reverse components of a reversible reaction.

[11] Neurospora crassa: Preparative scale for biochemical studies

Publisher Summary A variety of wild-type, auxotrophic, and morphological mutant strains of Neurospora crassa have been used for the preparation of enzymes, ribosomes, mitochondria, nuclei, cell walls, and other components of interest to the biochemist. Neurospora may be grown conveniently in liquid culture using 2.5–5-gallon Pyrex or Nalgene carboys. The medium is autoclaved for 40 minutes at 120° and 15 psi. Usually, sucrose is autoclaved separately for 20 minutes to prevent caramelization and is then added to the sterile medium. When strains of opposite mating type are grown on crossing medium, ascospores are formed in small sacs (asci) within the fruiting bodies (perithecia). Mature spores are ejected from the asci and can be collected by scraping the walls of the culture tube. Conidia, ascospores, and quick-frozen mycelia may be ground in a mortar and pestle with two volumes of acid-washed sea sand or alumina and one volume of buffer. When the preparation of intact mitochondria or nuclei is desired, lyophilized mycelia should not be used. Gentle grinding of fresh mycelia with a mortar and pestle and sea sand or alumina, or the use of a ball or grinding mill avoids disruption of cell organelles. Such methods have also been used for preparation of ribosomes although larger quantities of ribosomes with higher specific activity can be prepared from lyophilized mycelia.

[49] Tryptophan synthetase (Neurospora crassa)☆

Publisher Summary This chapter presents the assay, purification, and properties of tryptophan synthetase. The most commonly used assay is based upon the disappearance of indole. Indole, extracted with toluene, is measured spectrophotometrically at 540 mμ after reaction with Ehrlichs reagent. Indole-3-glycerol phosphate oxidized by periodate to indole-3-aldehyde is extracted with ethyl acetate and measured at 290 mμ. Glyceraldehyde-3-phosphate is determined readily with glyceraldehyde-3-phosphate dehydrogenase and nicotinamide adenine dinucleotide (NAD). One unit of enzyme is the quantity of enzyme that will convert 1 micromole of substrate in 1 minute at 37°. All purification solutions are prepared using deionized distilled water, and all operations are carried out at 2–4°. All pH measurements are made by the direct immersion of the electrodes into the solution, the pH meter being in the cold room. The purified enzyme is homogeneous as judged by sedimentation velocity and sedimentation equilibrium. The enzyme is sensitive to sulfhydryl reagents, such as mercuribenzoate, iodoacetamide, N-ethylmaleimide, and dithiobisnitrobenzoic acid. The loss in activity is accompanied by a loss of titratable sulfhydryl groups.

ANTIGENICALLY ACTIVE FRAGMENTS OF TRYPTOPHAN SYNTHETASE

The technique of enzymatically hydrolyzing proteins and examining the digests for serologically active fragments has been employed recently by a number of investigators (Porter, 1957, 1959; Morton and Deutsch, 1961; Cebra, 1961; Brown and Delaney, 1961; Kaminski, 1962; Metzger et al. 1962). This approach has been applied to the Neurospora tryptophan synthetase system for a number of reasons: ( 1) Sufficient quantities of antigen and antibody can be prepared at purities suitable for use in conventional immunochemical techniques; ( 2) rabbit anti-enzyme inhibits tryptophan synthetase enzymatic activity, a circumstance which greatly facilitates quantitative determination of antigen and antibody; ( 3 ) although tryptophan synthetase is too large for amino acid sequencing by present techniques, there exists the possibility of producing antigenically active fragments of the enzyme small enough for sequence determinations; ( 4 ) many tryptophan synthetase mutants of Neurospora crmsa produce a genetically altered protein ( CRM ) , serologically related to the wild type enzyme. It is possible that certain CRM’s may be structurally similar to some of the fragments in the digests of tryptophan synthetase; ( 5 ) antisera prepared against polypeptides purified from tryptophan synthetase digests may be helpful in the further study of CRM-less mutants ( Suskind, 1957); ( 6 ) such sera may also be useful in screening for specific sites (catalytic, antigenic, or cofactor binding) in both mutant and wild type enzymes, a technique which would be valuable in elucidating the specific structural alteration in certain CRM’s; ( 7 ) although Escherichia coli tryptophan synthetase does not exhibit cross reactivity with the Neurospora enzyme, many analogies between the two systems have been found (Suskind and Yanofsky, 1961). It is possible that cross reactivity studies with digests of Neurospora

Biochemical genetic studies of cycloheximide resistance in Neurospora crassa

Genetic analysis of a number of cycloheximide-resistant mutants of Neurospora crassa has shown that resistance is controlled by several genes. Two of these appear to be located on linkage group V. Resistance to the antibiotic is dominant in wild-type-mutant heterokaryons. Two types of cycloheximide-resistant mutants were isolated: one type exhibited colonial morphology only when grown in the presence of cycloheximide and the other type maintained normal morphology even at high concentrations of the antibiotic. Reconstitution experiments with supernatant solutions and 80S monosomes prepared from wild-type and resistant mutant strains indicated that the property of cycloheximide resistance most likely is associated with the ribosomes. No electrophoretic or serological differences were found between the ribosomal proteins of the wild-type and resistant mutants.