Jack-bean meal

Ltease test. The enzyme uretwe hydrolyses urea to ammonium carbonate (p. 519). The reaction is sp ific and is frequently used for solu tions of urea to which the biuret test cannot be applied. Add about 5 drops of phenohred to o 2 g. of urea dissolved in 5 ml. of water. To this yellow solution, add 0 2 g. of jack bean meal suspended in 2 ml. of water containing. also 5 drops of phenol-red. The colour changes to red as the solution becomes alkaline. 

Urease is one of the enzymes which have been obtained in the crystalline state. This has been done by stirring jack bean meal with 30°o aqueous acetone, filtering and allowing the filtrate to remain at o for several hours. The urease which crystallises out is separated by centrifuging and is then recrystallised. Like crystalline pepsin and trypsin, it is a protein. 

Place about 0 2 g. of jack-bean meal in a test-tube, add 2 ml. of water and about 5 drops of phenol-red. Mix thoroughly and allow the faintly yellow solution to stand while the urea solution is being made up. 

Urease solution. Place about 5 g. of jack-bean meal in a mortar and grind up with about 10 ml. of water, t hen add about 90 ml. of water, mix thoroughly and allow to stand for some time in order to deposit starch and other insoluble substances. Decant off the supernatant liquid into a conical flask and cork the latter. 

Historically the earliest Ni-containing enzyme to be described was urease from jack bean meal, which was crystallized by James Sumner in 19261. However, analytical techniques did not allow urease to be recognized as a Ni-containing enzyme until 50 years later. Urease catalyses the hydrolysis of urea to ammonia and carbamate, which spontaneously hydrolyses to give carbonic acid and a second molecule of ammonia. 

The protein nature of enzymes was established through the seminal work of James Sumner. In 1926, Sumner succeeded in isolating the enzyme urease in crystalline form from jack bean meal. This was the first time in history that an enzyme had been obtained in crystalline, though not completely pure, form. Subsequently, Sumner established that the crystalline enzyme was a protein. Urease is an enzyme that degrades one of the human end products of nitrogen metabolism, urea, to ammonia and carbon dioxide ... 

Jack-bean meal is a rich and convenient source of a-D-manno-sidase,34 and the enzyme displays zinc-dependence in characteristic fashion.38 At pH values below neutrality, the enzyme is unstable unless a zinc salt is added, and the instability is particularly marked during dialysis or gel chromatography. In our first method of purification,38 the later stages were performed at pH 5 in the presence of 100 yM zinc sulfate. Table V gives a brief outline of the purification procedure. 

Rat epididymis is also a good source of a-D-mannosidase, but it is not so readily available, in quantity, as jack-bean meal. A procedure similar to that just described was employed to obtain the enzyme from rat epididymis60 in the same state of purity as the jack-bean a-D-mannosidase, including freedom from /J-D-mannosidase and glycoaspartamidase. 

At pH values above neutrality, jack-bean a-D-mannosidase, unlike the enzyme from the other two sources, is stable, and the native, protein-metal complex does not dissociate. By working at pH 8, it is possible to purify the enzyme from jack-bean meal without addition of zinc.27 The procedure shown in Table V was followed with only slight modification (see Table IX, Section III,5 p. 433). 

As discussed under Purification and pH and Stability [see Sections 11,4 (p. 409) and 11,6 (p. 413)], a-D-mannosidase is unstable at low pH values unless Zn2+ is added. In the following experiments employing a partially purified, jack-bean meal preparation (see Table V, stage 3 p. 410) to which Zn2+ had not been added, the enzyme was pre-incubated at 37° and pH 5 before assay at the same pH with p-nitrophenyl a-D-mannopyranoside as the sub-... 

Fig. 4.—Effect of Zn2+ and EDTA on a-D-Mannosidase Activity85 in a Partially Purified, Jack-bean Meal Preparation Incubated for Various Periods of Time at 37° and pH5. [ , Enzyme alone , enzyme + 1 mAf ZnS04 andO, enzyme-1- 1 mMEDTA. The results are expressed as a percentage of the activity in the unincubated, enzyme preparation.39]...

Fig. 9.—Hydrolysis of 6 mM p-Nitrophenyl a-D-Mannoside39,95 by a Partially Purified, Jack-bean Meal Preparation for Various Periods of Time at 37° and pH 5.[, No addition in the presence of O, 100 /J.M ZnS04 , 100 (jM EDTA and A, 10 (iM CdS04.]...

To establish that a-D-mannosidase is a metalloenzyme, the proportion of bound zinc in the active molecule must first be determined. This experiment has been performed for the enzyme from jack-bean meal.27 Two types of preparation were employed. The first was purified, throughout, at pH 8 without any addition of Zn2+ (see Section 11,4 p. 409). The second enzyme preparation was purified in the presence of added Zn2+ at pH 5 up to stage 5 of the original procedure (see Table V p. 410). It was then freed from unbound Zn2+ by dialysis against glycine buffer of pH 8, and passed through a column of Sephadex G-100 in the same buffer. As may be seen from Table IX, the final specific activity was, in each instance, slightly less than that shown in Table V. 

In view of the isolation of the crystalline enzyme from this source (chosen because of the high concentration) 45 years ago, it might be supposed that a discussion of preparative procedures would be unnecessary. On the contrary, vigorous contentions concerning the purity and activity of crystalline urease have been a hallmark of its literature right to the present. Several factors contribute to this situation. The urease content of jack bean meal varies according to the origin and... 

As Mamiya and Gorin (11) note, Sumner occasionally had difficulty in preparing crystalline urease from some jack bean meals and this is a technical problem that has received attention in each recent improved method of preparation (11-13). Modification of Sumner s extraction procedure by including / -mercaptoethanol (11, 14) to diminish aggregation and ethylenediaminetetraacetate (EDTA) (13,15) to maintain a low concentration of metal ions has been helpful. The procedure used in this laboratory with consistent success over a period of 4 years, and involving several students, is essentially that of Mamiya and Gorin (11) but with EDTA included in the buffers. [There is an error in Mamiya and Gorin (11). The concentration of acetone used was 32%, not 36%.]... 

A method for the isolation of urease from hydrated jack beans developed by Sehgal and Naylor (15) employs DEAE-cellulose as a final purification step. A method intended for purification of urease from mammalian sources (16) was developed using jack bean meal as a standard source. It employed no acetone but is a combination of (NH4)2S04 precipitation and Sephadex column separations. [Note Fish-... 

The selective precipitation of some polysaccharides with antisera, which has been studied by Heidelberger and coworkers, is an important analytical tool, but the purification of preparative amounts of material by this method seems to be impractical. This could also be said of precipitation with the globulin from Jack-bean meal, concanavalin A, which has been used by Smith and his coworkers for fractionation. ... 

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