ANAI.YTICI\L BIOCnEMISTaY 203,47-52

(1992)

Spectrophotometric Ribonuclease Assays Dinucleoside Monophosphate Substrates

Using

Kimberley

K. Sandwick

Department

Received

M. Postek, of Chemistry,

Tracey

LaDue,

State Uniuersity

Cohn

of New

Nelson,

and

York at Plattsburgh,

Roger

Plattsburgh,

New York 12901

September 4, 1991

A pair of ribonuclease assays have been developed which offer improvements in specificity, simplicity, and/or sensitivity over current procedures. The assays measure the rate of adenosine release upon ribonucleas8 hydrolysis of 3’adenosyl dinucleoside monophosphate substrates. Adenosine formation is spectrophotometrically determined by combining a coupled-enzyme system (adenosine deaminase or an adenosine deaminaselnucleosidepbosphorylaselxanthiaeoxidasecombination) to the ribonuclease cleavage. As demonstrated by a brief characterization of the ribonuclease activities in several mouse tissues, the methods demonstrate the advantage of being able to discriminate between ribonucleases of differing substrate specificities. An interesting Buanosyl(3’-5’)adenosine-specific ribonuclease in mouse brain has been identified using these assay o 1882 ~~~~~~~~ P~~.~. nno. methods.

Nearly 2 decades after being the focus of many studies on enzyme kinetics and protein folding, ribonucleasas (RNases) are once again attracting great attention. This renewed popularity is driven much by the acknowledged crucial role that cytoplasmic RNases play in the control of cellular metabolism (1). The intricate balance between mRNA, poly(A)’ tails, RNases, and RNase inhibitors is gradually being sorted out, hopefully leading to an improved understanding of the process of RNA turnover and its effect on gene expression. Moreover, the discovery of extracellular “communicator RNA” (2) has given new meaning to the role of extracellular RNases found often in serum and elsewhere (3). These ’ Abbreviations used: RNase, ribonuclease; AD, nase: NP, nucleoside phosphorylase; X0, xanthine polycytidylic acid; ply(A), polyadenylic acid; XpA, cleoside monophosphate; UpA, uridyl~l(3’-5’)adenosine; lyl(3’-Y)adenosine; GpA, guanvlyl(3’-5’)adenosine; monophosphate.

adenosine deamioridase; poly(C), 3’.adenosyl dinuCgA, cytidyXp, 3’.nucleoside

RNases could eventually be assigned important regulatory roles in cell growth and differentiation. The greatly expanded list of different RNases now known to be present in tissues and fluids demands better assays for distinguishing among the individual members of the RNase group. Historically, the most popular method for RNase determination has been the Kunitz method (4-6). This procedure and modifications thereof (7-11) monitor the uv absorbance shift to shorter wavelengths of a hyperchromic RNA substrate upon enzyme hydrolysis. The methods are relatively simple, utilize an inexpensive substrate (often yeast RNA), and, although originally of poor sensitivity, now have reasonably good detection limits (in the nanogram range for ribonuclease A) (7,111. These assays suffer, however, from the inability to distinguish one RNase from another when together in a single solution. The hydrolysis rate observed thus represents a cumulative RNase activity. Precipitation and extraction techniques (12-15) offer improved sensitivity, but at the expense of greater complexity and longer assay times. In general, these methods are based on the solubility difference in strong acid or organic solvents of the smaller, hydrolyzed RNA pieces vs the larger, more intact RNA substrate. Like the Kunitz methods, the precipitation and extraction techniques measure cumulative RNase activity. In some cases, synthetic polynucleotides can be substituted to measure a particular RNase in the presence of other RNases. A common example is the use of a polycytidine (poly(C)) substrate for detection of a poly(C)-specific RNase in serum and tissue [see (12,16,17)]. A third common ribonuclease assay employs cyclic 2’,3’-pyrimidine nucleotides (typically cCMP) as substrate. These procedures make use of the fact that mammalian ribonucleases are “typically pyrimidinespecific” (18) and that the RNase mechanism includes cyclic 2’,3’-nucleotides as intermediates. These spectrophotometric (19,20) and titration (21) assays exhibit 47

48

POSTEK

improved specificity (they measure only pyrimidinespecific RNases) while being of somewhat lower sensitivity. Other procedures for measuring RNase activity include those whichuse high-pressure liquidchromatography (22,231, gel electrophoresis (24), or scintillation counting (25,26) for detection of reaction products. We describe below methods for RNase activity determination which offer some advantages over current procedures. The methods employ 3’.adenosyl dinucleoside monophosphate substrates: the choice of nucleoside at the 5’ end permitting the characterization of RNases of different substrate specificities. Dinucleoside monophosphates have previously been used as substrates in RNase specificity studies (see l&27). These studies determined RNase activity by monitoring the transient uv absorbance decrease resulting upon formation of a cyclic phosphate intermediate. (Reopening the cyclic nucleotide in the second step ofthe RNase mechanism subsequently reverses the absorbance back to its original value.) Due to its low sensitivity and its operation within the major protein absorbance region (at 286 nm), the procedure used in these studies has little application except when using purified enzyme. The assays reported here spectrophotometrically monitor the rate of adenosine formation from a dinucleoside monophosphate using a coupled-enzyme system. The procedures combine simplicity and short assay times with sensitivities approaching that of the precipitation/extraction methods. Most importantly, they provide a way to measure specific RNase activities present in a multi-RNase containing solution. EXPERIMENTAL

METHODS

Assay theory. The general assay format employs a dinucleoside monophosphate (XpA) substrate which, upon subsequent RNase action, results in a 3’.nucleoside monophosphate (Xp) fragment and adenosine.

XpA

RNes.

Xp

ET AL

WAVELENGTH (nm)

FIG. 1. Cal The UY spectrum of UpA (0.1 mu) and adenosine dearnnase prmr to (Curve 1) and subsequent to lCurve 2) RNase A addition. (b) The UY spectrum of UpA (0.1 rn~) and the three analytical enzymes IAD, X0, and NP) prior to (Curve 1) and subsequent to (Curve 2) RNase A additmn. The concentrations of reagents are those given in the assay protocols. The final spectra (Curves 2) represent complete RNase A hydrolysis of the dinucleoside monophosphate substrate.

the A,,, assay, RNase sine deaminase (AD) sine and ammonia:

XpA

=

Adenosine

Xp + H,O

is coupled with in the formation

adenoof ino-

+ Adenosine 3

Inosine

+ NH,

The initial rate of absorbance decrease at 265 nm (see Fig. la for uv spectra) is proportional to RNase activity. The A,,, assay couples the A,,, assay to two additional enzyme steps: nucleoside phosphorylase (NP) and xanthine oxidase (X0). Thus

XpA

+ Adenosine

Obviously, the ability of a particular RNase to cleave one of the XpA’s [i.e., guanosyl(3’-5’)adenosine (GpA), uridylyl(3’.5’)adenosine (UpA), adenosyl(3’-5’)adenw sine (ApA), and cytidylyl(3’.5’)adenosine (CPA)] will depend on the preference of that RNase for the substrate. For example, RNase A, a pyrimidine-specific RNase, will cleave CpA and UpA, but not GpA and ApA, RNase T , would preferentially cleave ApA (and at slower rates the other three dinucleoside monophosphates), and RNase T , would cleave only GpA, etc. The adenosine yielded by this cleavage is determined by linking the RNase reaction with additional enzymes and spectrophotometrically following the product(s). In

hydrolysis resulting

Inosine

s

Xp

Adenosine

+ H,O

+ P, %

Hypoxanthine

Hypoxanthine

+ 20,

+ Adenosine s

Inosine

+ NH,

+ Ribose-I-Phosphate ?%+ Uric

Acid

+ H,O,

The final product, uric acid, has a strong and relatively unique absorbance at 295 nm (see Fig. lb). Here, RNase activity will correlate with the initial rate of absorbance increase at 295 nm. In both procedures, the activities of the auxiliary enzymes (AD, NP, and X0) are incorporated at levels sufficiently high to make the RNase catalysis rate limiting.

RIBONUCLEASE

ASSAYS

DINUCLEOSIDE

I

I

RNase FIG. 2. strate.

USING

Sample

Weight

(pg)

The results of the A,, assay for RNase A with a UpA sub-

Mater&&. RNase A (type I-AS, bovine spleen), RNase T, (grade IV Asperigihs myzae), RNase T2 (grade VII, A. oryzae), adenosine deaminase (type IV, bovine spleen), xanthine oxidase (grade 1, buttermilk), nucleoside phosphorylase (calf spleen), uridylyl(3’-5’). adenosine, guanosyl(3’-5’)adenosine, and cytidylyl(3’5’)adenosine were obtained from Sigma Chemical Co. (Preparation and isolation of XpA substrates is possible by procedures involving micrococcal nuclease, RNA, and thin-layer chromatographic techniques (28).) Nucleoside phosphorylase, xanthine oxidase, and adenosine deaminase were purchased in ammonium sulfate suspension andused as such. The XpA solutions deteriorate slowly with time at 4°C and should be made fresh each day and/or frozen. A,,, assay. An assay mixture (2.0 ml) was produced containing 0.1 rn~ UpA and 2.5 units of adenosine deaminase in 0.05 M K,HPO, buffer (pH 7.2). The reaction was initiated by adding 50 pl of RNase solution to 2.0 ml of assay mix. The rate of absorbance decrease at 265 nm was determined. A, assay. One-tenth millimolar UpA, CpA, or GpA, 2.5 units of adenosine deaminase, 0.1 units of xanthine oxidase, and 0.64 units of nucleoside phosphorylase were combined in 2.0 ml of 0.05 M K,HPO, buffer (pH 7.2). The reaction was initiated by adding 50 ~1 of RNase (A, T,, or T,) and the absorbance at 295 nm was monitored. This assay was performed using both a chart recorder to measure the rate of absorbance increase and via a “fixed end-point” method where a single absorbance measurement at 295 nm was taken after 30 min of incubation at 22°C. Tissue studies. Tissue samples from the heart, liver, kidney, spleen, pancreas, and brain were removed from 3- to &month-old Mus musculus and quickly placed in 2-5 ml of ice-cold 0.05 M K,HPO, buffer (pH 7.2). The

MONOPHOSPHATE

49

SUBSTRATES

samples (typically 0.06-0.45 g in weight) were then homogenized for approximately 1 min using a Potter-Elvehjen homogenizer. The homogenized samples, kept on ice, were characterized for RNase activity within 2h following extraction using the As9, procedure. Homogenate sample sizes of 5-25 pl were used in a total assay volume of 1.0 ml (one-half scale assay). Tissue blanks were run by measuring an absorbance rate prior to sample addition. (A negative value represents a slower rate of absorbance decrease for the sample than that observed for the background.) Forcomparison,correspondingvaluesusingprecipitation techniques were obtained using procedures similar to those reported by Corbishley et al. (12). Briefly, yeast tRNA substrate (0.20 mg/ml) andtissue sample (100 al) were incubated at 37°C in 60 mM K2HP0, buffer (pH 8.0). Total assay volume was 0.50 ~1. Following 1 h of incubation, the enzyme reaction was quenched by addition of 0.50 ml of 1.2 M HClOJ0.022 M La(NO,), .6H,O. The solutions were placed on ice for 20 min and then centrifuged at 10,OOOg for 10 min. Four-hundred microliters of supernatant (holding the smaller, cleaved RNA pieces) was diluted with H,O to a 2.0-A volume and the absorbance read at 260 nm. Both tissue (no RNA added) and substrate (no tissue sample added) blanks were generated to correct the measured values. RESULTS A,,, assay. The results of the A,,, assay are shown in Fig. 2. The assay was linear (? = 0.989) in the RNase A sample concentration range, 0.003-0.04 mg/ml. The sensitivity of the assay was 3 pg RNase A/ml or 150 ng RNase A. The results of the A,, assay for RNase A A, assay. using a UpA substrate are shown in Fig. 3 (rate) and Fig.

f i

p

%

o.,* 0 IS 0.14 0.12 010 0.08 0.06 0.04 I 0.02 ~ 0.00 ! 0.0 0.1 RNase

FIG. 3. strate.

_’ 4 ,I,’ v’

0.2 Sample

0.3

04

Weight

The results of the A,,, amap for R&se

0.5 (pg)

A

with a UpA suh-

50

FIG. 4.

POSTEK

The results of the 30.min fixed end-point

AZ,, assay

4 (fixed end-point). The rate assay proved to be linear (? = 0.992) in the RNase A sample concentration range 0.0005-0.01 mg/ml. The sensitivity of the assay was 0.5 pg RNase A/ml or 25 ng RNase A. The fixed end-point assay proved to be linear (r’ = 0.9’78) in the RNase A sample concentration range 0.00006-0.001 mg/ml. The sensitivity of the assay was 12.5 ng RNase A/ml or 0.60 ng RNase A. Similar linear results were obtained for RNase T , and RNase T , (10 pl of sample in 1 ml total assay volume) usmg a GpA substrate ,n the A,,, rate assay and sample concentrations in the ranges 0.003-0.01 mg/ml for RNase T , and 0.005-0.01 mg/ml for RNase T,. For comparison, the rate of absorbance change per microgram RNase was 0.32 A min-’ pg-’ for RNase A (0.1 rn~ UpA substrate), 0.938 A mini’ pg~’ for RNase T , (0.1 rn~ GpA substrate), and 0.23 A mini’ pg-’ for RNase T , (0.1 rn~ GpA substrate). No activity was observed for RNase A using a GpA substrate or for RNase T , using a UpA substrate. Tissue studies. Table 1 gives the RNase activities of various mouse tissues using the A,,, procedure with CpA and GpA as substrates and the precipitation technique. One unit of activity in the A,,, assay and in the precipitation technique is defined as that activity which produces an absorbance change of 1.0 per minute of reaction time. The enzyme activities of the tissues were normalized on a weight of wet tissue basis. DISCUSSION The RNase assays presented are alternatives to the relatively insensitive Kunitz methods and the more lengthy precipitation techniques. The procedures are simple, contain no separation step, employ relatively stable reagents, and require (rate assays) 10 min or less to perform. The A,, assay, because of its greater absor-

ET AL. bance difference between substrate and product, exhibited a sixfold greater sensitivity vs the slightly more simple A,,, procedure. Whereas both methods proved acceptable in producing linear enzyme rate assays using purified RNase, the A,,, assay is better suited for characterizing the RNase activity of physiological Auids due to a relatively high native absorbance of these fluids at 265 nm. Improved sensitivity can be achieved by increasing the concentration of the substrate in the assay mixture. Our selection of a 0.1 rn~ XpA concentration was a compromise between satisfactory activity for the sensitivity desired and assay cost. Since we are operating at XpA concentrations far below K, [K, = 1.33 rn~ in our determination for RNase A using UpA substrate; similar values were obtained elsewhere (5,27)] an increase in the XpA concentration will directly translate into nearly proportional increases in assay sensitivity. The XpA assays offer several distinct advantages which make them attractive in certain situations. First, the RNase initial hydrolysis of the substrate should represent first-order enzyme kinetics with a linear absorbance change correlating to the number of cleavages. This is not true for the precipitation techniques where an indeterminate number of cuts of the RNA or polynucleotide substrate is required before any response in initial absorbance is achieved. The relatively small size of the substrate may be preferred in some circumstances [for example, see Ref. (2911. Second, the proper choice of dinucleoside monophosphate substrate allows greater specificity in characterizingparticular RNase activities contained in a given sample. In our mouse tissue study, a CpA-specific RNase was observed at extremely high activity levels in the pancreas. This is the mouse equivalent to the well-defined RNase A as based on its ability to specifically recognize pyrimidine bases on the 5’ side of the cleavage point. No RNase activity was observed in mouse liver by either the precipitation technique or our method. Previous studies (30) have reported that the human liver enzyme cleaves substrates yielding a purine on 5’ terminal end of the hydrolysis product (no major specificity on the 3’ terminal end). This implies that liver RNase should be capable of cleaving both CpA and GpA. Affecting both assays are ribonuclease inhibitors. Ribonuclease inhibitors (6,31), known to be present at relatively high levels in active tissues such as liver (31,321, brain (111, and placenta (20,33), have been shown to reduce the alkaline RNase activity of these tissues to negligible rates. Polyadenylic acid, existing as poly(A) tails of mRNA, has also been shown to inhibit RNases (34). We are continuing our investigation of the possible inhibition(s) of liver RNase via the A,,, assay using RNase inhibitor inactivators (sulfhydryl reagents like

RIBONUCLEASE

ASSAYS

USING

DlNUCLEOSlDE TABLE

Average

RNase CpA

Activity

Data of Various

Kidney Liver Heart Brain Pancreas SdeWl

AWE3ge Almin

for the A,,, and Precipitation

Tissues

Precipitation

A”%+T tissue

Units per gram

per gram tissue

sample weight (mg)

A”O& Almin

tmwe

of activity

f0.0012 -0.ow2

+1.23 -0.11

1.03 1.53

+0.0013 +0.OG05

+1.13 f0.32

1.07

+0.0010

+0239

0.67

+a0007

+o..so

0.46 1.44 0.60

+0.0004 +0.447 +0.0006

0.50 1.24 0.60

+O.W46 +a0039 to.0041

+9.1 f3.0 +6.8

in both

assays

is defined

as the

activity

needed

to generate

p-chloromercuribenzoate and Pb+*) and polyamines for poly(A) inhibition reversal. The RNase activity contained in the spleen was shown in our assay to be predominantly GpA specific. This could be either spleen phosphodiesterase (an exonuclease) (35) or a RNase generally categorized in the literature as the “liver-spleen” type. The fact that the spleen enzyme we characterized demonstrated no appreciable activity on a CpA substrate is interesting in light of the report by Bern& and Bern& (35) that spleen phosphodiesterase (from hog) fails to act on a poly(C) substrate. A GpA-specific rihonuclease activity of reasonably high rates was discovered in the brain. The precipitation technique [(17), for human brain] failed to detect RNase activity using RNA as substrate in this tissue. It is worth noting, however, that these procedures typally underestimate RNase activity in tissue samples of low RNase levels by yielding incorrect tissue blank absorbances (6). Radiolabeled RNA procedures (2526) are often employed to get around these problems. The GpA-specific activity we observe could be the relatively low, “free” (i.e., noninhibited) alkaline ribonuclease ativity observed in these latter reports (25), a 3’- or 5’-exonuclease (36,37), or some other previously unreported RNase in mowe brain. On the basis of its specificity, it is not the polyy(C)-specific plasma type as characterized in human brain by Neuwelt et al. (17). These spectrophotometric coupled-enzyme assays should find an appropriate place in the RNase researcher’s arsenal for sorting out the many types of alkaline RNase activities present in intra- and extracellular fluids. For example, we have found the CpA or UpA A,,, assay to be particularly useful for distinguishing pancreatic RNase activity from @dine-specific RNase activity in serum. (They both cleave poly(C).) Minor

tissue sample weight

0.96 1.63

to.91 +311.0 +1.0

assay

Average

Note. Weight of tissue and Almin normalized for a 15.~1 sample size. Duplicate performed in all cases except for the GgA assay of the heart sample. The precipitation unit

Assays

GpA substrate

Units

S3lllple weight (tug)

51

1

Mouse

substrate

AVWW? tissue

Tissue

SUBSTRATES

MONOPHOSPHATE

Units per gram

AW&W

(mg)

Almin

12.4 14.7

tissue

+0.0014 +0.0001

f0.11 +0.01

7.6

+0.0901

+0.01

4.3 9.9 4.3

0.0000 +0.010 +0.003

-0.07 +1.3 +I.6

trials on at least two different samples (n = 2-6) were data represents the average of five different trials. One

an absorbance

change

of 1.0

per

minute

(see

text).

modifications to the procedure (a two-step timed assay is required) will allow measurement of the acid RNase of lysosomes as well. Finally, the sensitivity and ease of operation of the XpA assays make them attractive for use in assessing the inhibition of RNases by RNase inhibitors and poly(A).

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