78 Extraction of Nitrate Reductase from Large Amounts of

Barley

The experiments described so far have all utilised small amounts of barley which could be homogenised quickly and efficiently by means of a mortar and pestle. However, the purification of spinach nitrate reductase reported by Notton, Fido and Hewitt (1977) utilised 1 kg of shoots and only yielded 0.4 mg of protein, indicating that to purify mg quantities of nitrate reductase several kilos of tissue must be used.

The efficiency of three extraction procedures was therefore evaluated:

(a) Extraction with a Mortar and Pestle

This was the most laborious and time-consuming method, but consistently gave the best results. When tissue was ground in Buffer I (3 ml buffer per gram tissue) and the ' cell debris removed by filtration through a double layer of muslin prior to centrifugation, high levels of both nitrate reductase activity and protein were obtained.

(b) Mechanical Homogenisation with a Waring Blender This method of extraction was found to be unsuitable for use with barley shoots. Due to the thin shape of the leaves and their small size (4 cm when nitrate reductase activity is greatest - see later) large numbers of shoots remain uncut. Also, due to the high speed of the blades.

a significant amount of frothing could not be prevented resulting in dénaturation of extracted proteins. No nitrate reductase activity could be detected in extracts prepared this way.

(c) Liquid Nitrogen - Mediated Cell Breakage Followed by Vigorous Mixing with Buffer I

In this method, shoots were harvested and then frozen

by addition of liquid nitrogen. The brittle shoots result­ ing from this treatment were then crushed to a powder in a

(

large mortar to which was then added the required amount of Buffer I (3 ml buffer per gram tissue). This 'soup' was then vigorously mixed with a large pestle and then stirred at 4®C for 30 minutes.

A direct comparison of the efficiency of methods (a) and (c) is given below in Table 4 in which 400 g of tissue was subjected to each method of extraction, cell debris

removed by filtration through a double layer of muslin and the nitrate reductase and protein levels measured in the resulting filtrates.

TABLE 4

(a) 23.42 7134

(c ) 1 2 . 8 8 1 4 6 1 3

* , ^

80

It is evident from these results that method (a) (mortar and pestle) is much more efficient in the extrac­ tion of nitrate reductase activity and protein than is method (c) (liquid nitrogen-assisted). A further problem encountered with method (c) was that subsequent centrifu­ gation resulted in precipitation of almost 50% of the nitrate reductase activity. In all subsequent work,

therefore, barley shoots were extracted by method (a) using a mortar and pestle.

Streptomycin Sulphate Treatment

As indicated earlier (Section II), treatment of a

barley extract with protamine sulphate, at the concentra­ tion suggested by Solomonson (1975), resulted in significant losses of nitrate reductase activity. An alternative method of removing nucleic acid material from the extract was suggested by Notton, Fido and Hewitt (1977) and involved

the use of streptomycin sulphate. Streptomycin sulphate was added to the filtered extract (5 mg streptomycin sul­ phate per gram of tissue extracted) prior to centrifugation and the pH restored to 7.5 if necessary by the addition of dilute NaOH. After stirring at 4°C for 10 minutes, nucleic acid material and cell debris were removed together by

centrifugation at 38-40 000 g for 50 minutes, the resultant supernatant containing the nitrate reductase activity.

During this centrifugation, approximately 30% of the protein precipitated. Nitrate reductase activity was some­

times found to increase at this stage by as much as %

but an several occasions was found to decrease, exceptionally by as much as 20%. The reasons for the variability found

at this, and other stages of the purification, will be examined in the Discussion at the end of this Chapter.

Ammonium Sulphate Fractionation

In early experiments (Section II), (NH^ÏzSO^ treat­ ment was used solely as a means of concentrating the pro­ tein samples for subsequent chromatography. It is likely, however, that a careful choice of (^#4 ) 2 8 0 4 concentrations

would itself provide some purification of the enzyme in addition to functioning as a concentration stage. A 50 g sample of barley shoots was therefore harvested, nitrate reductase extracted by grinding the shoots with Buffer I in a mortar, and cell debris removed by centrifugation.

A series of (NHi^)2S0 i+ fractionations was then carried out

on the extract at (#5 4 )2 8 0 % concentrations of 30%, 45%,

60% and 80%. At each stage, the solution was stirred for 30 minutes at 4®C and precipitated protein then collected

by centrifugation at 2 0 0 0 0 g for 2 0 minutes, dissolved in

10 ml of Buffer I and then assayed for nitrate reductase activity (Fig. 16).

The bulk (67.8%) of the nitrate reductase activity was found to precipitate between 30% and 45% saturation with (#5 4 )2 8 0 % (Fig.16) while a further 18.9% was preci­

FI G . 16

(NHu)9SQ% Precipitation Characteristics of

Barley Nitrate Reductase

This figure illustrates the required (#5 4 )2 8 0 %

concentrations for precipitation of barley nitrate reductase. Experimental details are given in the main text.

a» ta 80 - 70 — •S 60_o s 50 S 40 ai OC 30 5 20 s? 10 0 10 20 30 40 50

%ESEÜ1

82