EL DEPORTE EN LA COTIDIANIDAD DE LOS ADOLESCENTES

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started there was very little information available in

the literature concerning the sizes and structures of higher plant nitrate reductases. Estimates of molecular weight varied greatly from about 160 000 (Hageman and Hucklesby,

1971) to about 500 000 (Anacker and Stoy, 1958) with an apparent average value of about 300 000 (Jolly, Campbell and Tolbert, 1976). Notton, Hewitt and Fielding (1972) determined a value of 240 000 for spinach nitrate reduc­ tase by gel filtration through Biogel AO.5 m whilst the same authors (Notton, Icke and Hewitt, 1976) reported a value of 152 000 for the same enzyme determined by sucrose density gradient centrifugation. This diversity in values for the same enzyme illustrates the difficulty in inter­ preting the data reported in the literature and stems

mainly from the fact that all these authors have used only one technique for the determination of molecular weight. For this to be accurate nitrate reductase would have to be a perfectly globular protein which behaved ideally under all test conditions. The conflicting values reported for the molecular weight of spinach nitrate reductase indicate strongly that this is not an ideal protein. Hence any

conclusions regarding the structure of higher plant nitrate reductase based on these results must be regarded with

caution.

A more reliable and accurate method for the deter­ mination of the molecular weight of a protein, even in

Rather than relying on only one measurement, this method involves the determination of both the sedimentation coefficient of the protein by sucrose density gradient centrifugation and the Stokes radius of the protein by gel filtration. The molecular weight of the protein can be calculated from this data, by means of the equation given in Methods Section IV, together with an indication of the shape of the protein. It is this method which was used throughout the work to be reported in this thesis.

Although no higher plant nitrate reductase had been characterised this way prior to the start of this work, the Siegel and Monty (1966) method had been used to deter­ mine accurate molecular weights for the nitrate reductases

from Chtovella (= 356 000 - Solomonson et a l., 1975), E. crassa (= 228 000 - Garrett and Nason, 1969) and

A. nidulans (= 190 000 - MacDonald and Coddington, 1974).

It was therefore clear that there were significant differences in the sizes of the nitrate reductases from these sources

despite the great similarity between the components of the enzyme (see part (3) of this Introduction) but the reasons for these and their significance in terms of the structures of the enzymes was not clear.

«

During the past three years our understanding of the nitrate reductases has greatly improved and current ideas and models for the structure of these enzymes will be

presented in the General Discussion where they will be

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throughout the course of this work.

6 . Biochemical Genetics of the Nitrate Reductases (a) A, nidulans

Early genetic work with A. nidulans (Cove and Pateman, 1963; Pateman et al., 1964) revealed the presence of six unlinked genes in which mutation resulted in loss of the ability to grow on nitrate as sole nitrogen source.

Mutation in any of five of these genes (designated cnx) v;as also shown to result in loss of the ability to grow on

hypoxanthine and it was proposed (Cove and Pateman, 1963) that the cnx genes coded for a cofactor which was common to both nitrate reductase and xanthine dehydrogenase. One of the cnx mutations {cnx E) was subsequently shown

(Arst, MacDonald and Cove, 1970) to be repairable by

growth in the presence of high concentrations of molybdate indicating that the cnx E gene product is likely to be responsible for the insertion of Mo into nitrate reduc­ tase.

The sixth gene found by Pateman et al. (1964) to be essential for growth on nitrate was designated nia D and was believed to be the structural gene for nitrate reduc­

tase. This was confirmed by MacDonald and Cove (1974) who showed that a temperature-sensitive mutation in nia

D resulted in the production of a temperature sensitive nitrate reductase. Similarly, cnx H was shown to code for

a structural component of nitrate reductase whereas cnx E and onx F did not. Temperature-sensitive mutations at the other cnx loci {cnx ABC and cnx G) were not obtained and so could not be analysed.

Based on these genetic studies, MacDonald, Cove and Coddington (1974) proposed that A. nidulans nitrate reduc­ tase was composed of two nia D gene products held together by a Mo-containing cofactor which was coded for by the cnx genes.

(b) E. crassa

Genetic work with Æ. crassa has not been so detailed as with A. nidulans but nevertheless five loci (designated nit-1 to nit-5) have been identified as essential for

growth on nitrate as sole nitrogen source. In a series of papers (Sorger, 1965, 1966; Sorger and Giles, 1965) it was shown that two of these genes, nit-1 and nit-3 coded for structural components of N. crassa nitrate

reductase. The nit-1 gene was thought to code for a poly­ peptide which was responsible for the transfer of electrons

from Mo to nitrate (analogous to the product of the onx genes of A. nidulans) whilst the nit-3 gene was thought to code for the rest of the nitrate reductase molecule

(analogous to the product of the nia D gene of A. nidulans)

These observations were confirmed and extended (Nason et dl,, 1970, 1971; Ketchum et al., 1971) by the

15

demonstration that nitrate reductase activity could be reconstituted in vitro by the mixing of extracts from nit-1 and nit-3 mutants. Subsequent work (Nason et al. 1974) demonstrated that the nit-3 extract could be replaced by a Mo-containing component (MCC) derived by acid treatment from any of several Mo-containing enzymes tested. It could therefore be concluded that the nit-1 gene specifies a Mo-containing component which is common to several molybdoenzymes and is analogous to the cnx genes of A. nidulans. It is, however, surprising that only one nit-1 gene has been identified as there are

five cnx genes required in A. nidulans for the manufacture of MCC which has been shown (Lee et a l . , 1974) to be

dialysable, to have a molecular weight of only about 1 0 0 0 and to require added Mo for activity. It was therefore proposed by these authors that N. crassa nitrate reduc­ tase was composed of nit-3 gene products bound together by the small nit-1 gene product (MCC). This model is identical to that proposed for A. nidulans nitrate reductase by MacDonald and Coddington (1974).

(c) Algae

The most studied algal nitrate reductase is that from

Chlorella but no genetic analysis has been undertaken with

this species. However, two groups have reported genetic studies with another green alga, Chtamydomonas reinhardii Both groups (Nichols and Syrett, 1978; Nichols, Shehata

and Syrett, 1978; Sosa, Ortega and Barea, 1978) reported the existence of mutations analogous to the cnx {nit-1) and nia D {nit-3) mutations of A, nidulans {N. crassa) but insufficient data is available to allow definite conclusions to be drawn.

(d) Higher Plants

Genetic studies are made difficult in higher plants by the fact that these are not haploid and so any recessive mutation is masked. Despite these problems, nitrate-

reductase-negative mutations have been obtained in

Nicotiana tabacum (Müller et al., 1976) and some of these have been shown to also lack xanthine dehydrogenase activity

(Mendel and Müller, 1976) providing evidence that these enzymes share a common cofactor in higher plants as well as in fungi.

Mutants lacking nitrate reductase activity have also been obtained in barley (Warner, Lin and Kleinhofs, 1977) but insufficient data was reported to allow these to be evaluated.

7. Determination of the Physiological Reductant of Higher