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N. J. Microbiology, 2(2), 1982, pp 195 - 2 0 6

© The Nigerian Society f o r Microbiology 1982.

PARTIAL CHARACTERIZATION OF A TRYPSIN STIMULATED

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NIGERIAN J O U R N A L OF MICROBIOLOGY, VOL 2 , NO 2, 1982 MATERIALS AND METHODS

Chemicals

Nucleotides were products of p-L Biochemicals, Inc. Milkwaukee, Wisconsin, or of Sigma chemical company, Saint Louis, Missouri. Dicyclo-hexyl-carbodiimide (DCCD) was purchased from Eastman Kodak C o m p a n y , Rochester, New York. All other chemicals used were either ragerit or A.C.S. grade.

Bacteria

Virulent Agrobacterium tumefaciens strain C-58 was obtained f r o m Dr. Mary-Dell Chilton of the University of Washington in Seatle, Washington. The virulence of C-58 was confirmed throughout t h e period of this investigation by inoculation of stem tissue of life plant, Kalan-choe daigremontiana and Tobacco, Nicotiana tabacum. T u m o r tissues developed at sites of needle puncture a f t e r 14 days of incubation at 28°C.

Growth Conditions

The bacteria were cultivated at -26°C on a shaking incubator in a liquid dextrose 0.5% basal medium enriched with 1% yeast extract, 0.5% sodium chloride and 1% Peptone as previously described by Hamilton and Fall (1971). The bacteria were grown in a 2-liter Erlenmeyer falsk containing 1.0 litres of the medium described above.

Exponential phase cells were harvested b y centrifuging at 17,000 x g.

Extraction of Enzyme

Twenty g of cells were cracked b y a m o r t a r and pestle, using alumina as abrasive in 20ml of IMTRIS 2mM M g+ + at p H 7.5 and t h e mixture subjected to centrifugation at 3 0 , 0 0 0 x g f o r 1 hour. A buffer of this high ionic strength will prevent the release of the membrane bound ATPase, which is tightly bound to t h e membrane and could only be released by low ionic stren-gth buffers. The supernatant fluid f r o m the above centrifugation was the source of <he cyto-plasmic soluble ATPase.

Enzymatic Assay

ATPase activity was assayed b y colorimetric measurement of inorganic phosphate (Pi) released from ATP, by the method of Fiske and Subbarow (1925)as modified b y Dulley (1975).

The reaction mixture ( I m l ) contained 0.1ml of 2.5 x 10"2M nucleotide, 0.3ml (70 - 100 ug protein) enzyme source and O.frnl of 0.1M Tris-ECI (pH 7.5').

When the effects of cations and k n o w n inhibitors were tested, 0.1ml of the agent was pre-incubated with t h e reaction mixture. Final volume of t h e reaction mixture was always adjusted to 1 ml with t h e b u f f e r . The mixture of enzyme and b u f f e r was pre-incubated for 10 min at 3 7 ° before t h e addition of substrate, and t h e incubation was continued for 30 min at 3 7 ° C iq, a water b a t h shaking incubator.

A control mixture contained all t h e c o m p o n e n t s of t h e test medium exoept t h e enzyme source so as to correct f o r t h e endogenous breakdown of t h e substrate.

Protein concentration was determined by t h e m e t h o d of Lowry et al. (1951), or by U.V.

absorption.

Enzyme Purification

All operations were carried out at 2-5°C as follows: The cytoplasmic soluble fraction (obtained as described above) was applied on a DEAE-Sephadex A-50 column and fractions were collected b y elution with a stepwise gradient using Tris b u f f e r s in t h e following order:

0.1M Tris-HCl + 5mM Mg*4" PH 7.5 , 0.0M Tris-ECI + 5mM M g+ + PH 7.5 and 0.3M Tris-HCl

SHONUKAN - A TRYPSIN-STIMULATED ADENOSINE TRIPHOSPHATASE 197

+ 5mM Mg44" pH 7.5 respectively. Active fractions wrer n o t eluted until a f t e r t h e application of buffer of 0.3M Tris-HCl + 5mM M g ^ pH 7,5. Pooled active fractions were used in t h e s t u d y of thfc enzyme without f u r t h e r t r e a t m e n t . F o r e l e c t r o p h o r e t i c studies, t h e e x t r a c t was concentrated 10-fold with dry sephadex G-25 without any loss of activity. A m m o n i u m sulphate concentra-tion, on t h e other h a n d , inhibited the activity of t h e enzyme. As a result of the n o t e d inhibi-tory action of ammonium sulphate, t h e salt was n o t used t o concentrate t h e e x t r a c t b e f o r e application onto t h e DEAE — Sephadex column.

A unit of enzyme is the amount of enzyme that releases 1 u mole of inorganic p h o s p h a t e in 30 min.

Sodium Dodecyl sulfate (SDS) Gel Analysis

SDS gel analysis was performed b y the m e t h o d of Laemmli and King (1971).

Effect of Physico-Chemical Conditions on enzyme activity:

Effect of various c o m p o u n d s

Enzyme activity was assayed u n d e r standard conditions exoept that t h e following sub-stances at the indicated concentrations were added. 2,4 Dinitrophenol ( l x l O "3 M), Sodium azide ( l x l O "3 M), Dicyclo-hexylcarbodiimide ( l x l O "2 M), Ethylene diaminetetra-acetic acid ( l x l O "3 M), Potassium fluoride ( l x l O "3 M), and Trypsin (500 microgram): The activity of t h e standard reaction mixture was taken as 100%.

Stability of enzyme at selected temperatures

Samples of cytoplasmic ATPase preparation obtained b y DEAE — Sephadex chromatogra-phy were stored at 4°C, 2 8 ° C and 3 7 ° C respectively. At intervals aliquots were w i t h d r a w n and the activity was assayed under t h e standard conditions.

Effect of cations on enzyme activity

Enzyme activity was assayed u n d e r standard conditions e x c e p t that t h e reaction mixture was made 10 micromole with respect to t h e added cations. The cations tested were Mg"1"1", M n+ +, Ca++, Zn4"1" and Ni"1"1

"-Effect o f m e r c u r i c chloride ( H g c l 2 ) on enzyme activity

Enzyme activity was assayed u n d e r standard conditions e x c e p t that t o t h e reaction m i x t u r e was added 1 x 1 0 "2M H g c l 2 .

Effect of pH on enzymatic activity

Activity was measured under standard conditions except that 50mM buffers (Acetate — NaOH f o r pH below 6, Tris-HCl, pH 6.0 - - 8 . 0 , and glycine - NaoH pH 9.0 - 1 2 . 5 ) were used!

Percentage activity was computed using highest activity as 100%.

Effect of incubation temperature on enzyme activity

Ezyme activity was assayed u n d e r standard conditions except that four d i f f e r e n t tempera-tures (4°C, 28°C, 37°C, and50°C) of incubation were checked.

RESULTS

Enzyme Fractionation

The chromatographic profile of the cytoplasmic soluble ATPase on DEAE-Sephadex A-50 showed t w o major peaks of activity (Fig. 1), which were eluted with 0.3M Tris b u f f e r as

indica-15,8

NIGERIAN J O U R N A L OF MICROBIOLOGY, VOL 2, NO 2, 1982

ted. Peak I was used for work reported here.

The enzyme was about sevenfold purified by DEAE-Sephadex A-50 fractionation (Table I).

The pooled active fractions from DEAE-Sephadex step lost all activity when subjected t o cellex-D fractionation.

Effect of physico-chemical conditions on the activity of t h e enzyme.

Known inhibitors of members ATPase including potassium fluoride, Dinitrophenol, Dicyc-lohexyl-carbodiimide (DCCD), sodium azide and ethylene diamine tetracetic acid

(EDTA) had n o effect on the activity of the enzyme (Table 2). However, trypsin activated the enzyme 31% above the control level.

With respect to the stability of the enzyme at different storage temperatures, less suscep-tibility to inactivation at 4 «c t h a n a t o t h e r temperatures was observed (Fig. 2). The reason f o r the instability of the cytoplasmic soluble ATPase at 2 8 ° C and 3 7 ° C is n o t k n o w n at present but could b$ due t o a natural inhibitor of soluble ATPase and/or contaminating proteases that are active at these temperatures.

The enzyme was slightly stimulated by all the divalent cations tested (Table 3).

Mercuric chloride (HgCI2) at a concentration of 1 x 10/ 2M inhibited the cytoplasmic solu-ble ATPase by 34%.

The pH o p t i m u m of activity f o r the cytoplasmic soluble ATPase was f o u n d to b e between 8.0 and 9.0 (Fig. 3). When different incubation temperatures were checked, the o p t i m u m tem-perature of activity was found between 28 and 50°C (Fig. 4). When aliquots of the sephadex G-25 concentrated enzyme were subjected t o SDS-polyacrylamide gel electrophoresis, poly-peptide chains of molecular weights of 30,000, 60,000 and 200,000. Daltons were f o u n d by comparison with standard marker proteins (Fig. 5).

Table 1:

Summary <jf Purification of Cytoplasmic Soluble ATPase form c-58 Strain of Agrobacterium tumefacines

Steps

Total Protein, mg

Total Activity Micromoles Pi

(releasedI30 min)

Specific Activity Micromoles Pi

(released/30 min/mg Protein)

Crude

-extract 196 149.9 0.765

DEAE

-Sephadex- A-50 40 233 5.84

SHONUKAN - A TRYPSIN-STIMULATED ADENOSINE TRIPHOSPHATASE 199

TABLE 2:

Effect of Various Compounds on Cytoplasmic Soluble ATPase Activity Values represent average of triplicate assays.

Micromoles PO4 Relative Compound Added Released Activity, %

None 0.875 100

1X10 3M 2,4 DNP 0.850 96

1X10-3 NaNj 0.850 96

1 X 1 0 " ^ DCCD 0.875 100

1X10"3 EDTA 0.850 96.9

1X10"3MKF 0.825 94

500 ug Trypsin 1.15 131

* 2,4 DNP - 2,4 Dinitrophenol NaNj - Sodium azide

DCCD - Dicyclo4iexyl-caibodiimide EDTA - Ethylene diamine tetra-acetic acid KF — Potassium Fluoride

TABLE 3:

Effect of Divalent Cations on Cytoplasmic Soluble ATPase Activity.

Values represent average of triplicate assays.

Relative

Cation Micromoles Activity PO4 %

None 1.35 100 lOmMMg** 1.40 103

lOmMMn*"1" 1.40 103

lOmMCa4"1' 1.50 111

lOmMZn** 1.50 111

lOmM Ni4"4 1.45 107

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NIGERIAN J O U R N A L OF MICROBIOLOGY, VOL 2, NO 2 , 1982

DEAE-Sephadex Extraction of