A comparative study of hepatic and epidermal histidase in the guinea-pig (cavia porcellus)

Comp. Biochem. Physiol. Vol. 84B, No. 4, pp. 523-529, 1986

0305-0491/86 $3.00 + 0.00 Pergamon Journals Ltd

Printed in Great Britain

A COMPARATIVE STUDY OF HEPATIC A N D E P I D E R M A L HISTIDASE IN THE G U I N E A - P I G

(CA VIA PORCELLUS) ROGER L. ALLEN, ROGER HOPEWELL and COLIN PROTTEY* Department of Chemistry and Biochemistry, Liverpool Polytechnic, Liverpool L3 3AF, UK; and *Unilever Research Laboratory, Port Sunlight, Wirral, Merseyside L62 3JW, UK

(Received 9 August 1985)

Abstract--1. Histidine ammonia lyase was purified to homogeneity from guinea-pig liver and epidermis. 2. Both enzymes had similar molecular weights, subunit composition and pH optima. 3. Km values for the two were similar at pH 9.2 but different at pH 7.0. 4. Both enzymes were stimulated by low thiol concentrations and inhibited at higher concentrations, but to different extents. 5. Antibody to the hepatic enzyme showed complete identity against hepatic enzyme but incomplete identity against epidermal enzyme.

INTRODUCTION Histidine a m m o n i a lyase, or histidase (EC 4.3.1.3) is present in both liver and epidermis of mammals (Schwartz, 1961; Zannoni and LaDu, 1963). The developmental pattern of the epidermal and hepatic enzymes differs in mammals, histidase being present in the epidermis but not in the liver at parturition (Baden et al., 1968). During puberty hepatic histidase activity increases, while in the epidermis enzyme activity decreases (Feigelson et al., 1976). It has been sugested that the epidermal and hepatic enzymes in the rat are isoenzymes on the basis of these developmental differences and on differences in kinetic behaviour of the enzyme from the two tissues (Baden and Gavioli, 1974). However, other studies suggest that the two rat enzymes are in fact identical, in so far as they have identical kinetics and appear immunologically identical (Bhargava and Feigelson, 1976). We have attempted to resolve this apparent diversity of view by examining the kinetics and immunological identity of highly purified samples of hepatic and epidermal histidase, from the guinea-pig

( Cavia porcellus ).

EXPERIMENTAL

Animals Adult female guinea-pigs (Cavia porcellus) of the Duncan Hartley strain were obtained from David Han Ltd., Darley Oaks Farm, Burton-on-Trent, Derbyshire. Antibodies were raised in adult male New Zealand White rabbits obtained from Hyline Rabbits Ltd., Northwich, Cheshire. All animals were given free access to both food and water.

Materials Bovine serum albumin, Coomassie brilliant blue G250, 1,4-dithio-L-threitol and basic fuchsin were purchased from

Sigma (London) Chemical Co.; blue dextran, Sepharose 6B, DEAE Sephadex A50, QAE Sephadex A50 and Sephadex G-200 (Superfine) from Pharmacia Ltd.; Freunds complete 523

adjuvant from Grand Island Biological Co. (USA) and sterile 'water for injection' from May and Baker Ltd. All other reagents were of Analar or equivalent grade.

Purification of hepatic histidase Livers from animals freshly sacrificed by cervical dislocation were washed in 0.9% sodium chloride solution, then dried, cut into small pieces and homogenised in 0.25 M sucrose at 4°C (1 g liver/2cm3 sucrose solution). The homogenate was centrifuged at 100,000g for 60min (all centrifugations were carried out in a MSE 65 ultracentrifuge using an 8 x 50 cm 3 rotor, precooled to and run at 4°C). The supernatant was then purified by a modification of the procedure of Brand and Harper (1976). Supernatant was heated in thin-walled glass tubes for exactly 3 min at 65°C after which it was rapidly cooled in ice. All further operations were carried out at 4°C. Precipitated protein was removed by centrifugation at 45,000 g for 20 min and discarded. The supernatant was adjusted to 45% saturation with ammonium sulphate solution, saturated at room temperature and cooled to 4°C (the same ammonium sulphate solution was used in all subsequent fractionation steps). The precipitate was removed by centrifugation as before and the supernatant was adjusted to 50% saturation with ammonium sulphate, followed by continuous stirring for 20 min. The precipitate, containing most of the histidase activity was harvested by centrifugation as before, dissolved in 5 cm 3 of 0.1 M Tris-HCI buffer, pH 7.6 containing 0.1 M sodium chloride and loaded onto a column (30 x 3 cm) of DEAE Sephadex, pre-equilibrated with the same buffer. The column was eluted with the loading buffer and fractions containing the highest specific activity of histidase were pooled and incubated for 15min with dithiothreitol (2 mg/cm3 of solution). This solution was then subjected to a repeat of the ammonium sulphate fractionation described above. The final histidase-containing precipitate was dissolved in 2 cm 3 of 0.1 M Tris HCI buffer, pH 9.2 and loaded onto a pre-equilibrated column (65 x 1.5 cm) of Sephadex G-200 (Superfine). The column was eluted with the loading buffer and histidase-containing fractions were pooled. Histidase was precipitated by adjusting the pooled fractions to 60% with ammonium sulphate. This precipitate was dissolved in 2cm 3 of 0.1M Tris-HC1 buffer, pH 6.8 and loaded onto a pre-equilibrated column (25 x 2.5 cm) of diethyl ( - 2 hydroxypropyl) amino ethyl (QAE) Sephadex.

524

ROGER L. ALLEN et al.

Histidase was eluted by a linear gradient of sodium chloride (0 to 0.7 M) in the loading buffer. Fractions containing histidase with constant specific activity were pooled and stored at - 2 0 ° C for up to 6 weeks with no loss of activity.

Purification o f epidermal histidase Pelts from freshly sacrificed animals were quickly removed by dissection and were immediately depilated by clipping followed by shaving with an electric razor. The depilated pelts were then frozen on glass plates, dermis side down and the epidermal layer was removed by scraping with a blunt scalpel. Pooled epidermis from 10 to 20 skins was homogenised in a vortex homogeniser (Ultra-Turrax) in 100cm 3 of phosphate-buffered saline (0.8% sodium chloride, 0.02% KHzPO 4, 0.14% Na2HPO4), containing 33/~M zinc acetate and 0.1% Triton-X-100. The resulting homogenate was purified in a similar manner to that used for the hepatic enzyme, except that the initial heating treatment was found to denature the epidermal enzyme and was omitted. The epidermal enzyme was found to precipitate at 45% saturation with a m m o n i u m sulphate; the supernatant from the initial homogenate was therefore brought to 38% saturation. The precipitate was removed by centrifugation as before prior to precipitating the histidase at 45% saturation. This fractionation was repeated after D E A E Sephadex chromatography as with the hepatic enzyme. The remainder of the purification procedure was then identical to that described above. Assay for histidase Histidase activity during purification was assayed by a method similar to that of Mehler and T a b o r (1953). The assay mixture, containing L-histidine mono-hydrochloride (3.3 mM), reduced glutathione ( l . 6 6 m M ) and Tris HC1 buffer, pH 9.2 (0.1 M), in a total volume of 2 . 9 c m 3 was maintained at 37°C. Reaction was initiated by addition of 0.1 cm 3 of enzyme extract and product formation was monitored continuously at 2 7 7 n m in a Pye Unicam SP1800 uv/visible spectrophotometer. The concentration of urocanate produced was calculated from the molar extinction coefficient of 18.8 m M ~ cm t and enzyme activity was thus expressed as the increase in urocanate concentration per unit time. For most of the kinetic studies a different assay was used to allow for a greater number of replicates. The assay mixture, containing L-histidine mono-hydrochloride (0-4.0 mM), reduced glutathione (1.66 m M ) and T r i s - H C l buffer, pH 9.2 (0.02 M), in a final volume of 2.4 cm 3, was incubated for 15 min at 37°C and the reaction was initiated by the addition of 0.1 cm 3 of enzyme solution. The incubation was continued for between 1 and 4 hr (or as long as the increase in the concentration of urocanate was linear) and the reaction was stopped by the addition of 0.5 cm 3 of 2 M perchloric acid. Precipitated protein was removed by centrifugation at 3000 g in a Hettich maxi-fuge centrifuge. The optical density of the supernatant was measured at 266 n m (266 n m is the 2max in acid conditions; molar extinction coefficient is unchanged). Separate controls lacking enzyme and histidine were treated similarly and their optical densities were subtracted from the test samples before calculation of urocanate concentration as before. Histidase activity was again expressed as concentration of urocanate produced/minute. Determination o f K m Epidermal and hepatic histidase activities were assayed as above using a concentration range of L-histidine from 0.01 to 1.0 m M . K m values were extrapolated from Lineweaver Burk double reciprocal plots subjected to linear regression analysis. Protein determination Protein concentration was normally assayed by the

method of Sedmak and Grossberg (1977). However, during purification of the enzymes, the protein concentrations were determined (after dialysis if necessary) by the Folin Ciocalteu procedure (Lowry et al., 1951) using bovine serum albumin as a standard.

Polyacrylamide gel electrophoresis Discontinuous polyacrylamide gels (140 × 140 × I ram) were prepared by the method of Lamb et al. (1976) both in the presence and absence of sodium dodecyl sulphate (SDS). Electrophoresis was at constant voltage either at 200 V for about 3 hr or 40 V for about 16 hr. Gels were stained with Coomassie brilliant blue G250 using the method of Holbrook and Leaver (1978).

Production o1 antibodies 2 0 0 # g of purified enzyme was dissolved in 0 . 5 c m -~ of pyrogen-free "water for injection", to which was added 0.5 cm 3 of Freunds complete adjuvant. The mixture was emulsified. A 3 kg rabbit was bled from an ear vein to obtain control serum and was then injected intramuscularly with the emulsion. A booster injection, prepared as before, was administered after 3 weeks and blood was collected after a total of 5 weeks by cardiac puncture. Alternatively, further booster injections were administered after 5 and 7 weeks and blood was collected after a total of 10 weeks. Blood was allowed to clot for 60 rain at room temperature and was left overnight at 4 C . Serum was collected after centrifugation at 2000g for 10min in an MSE Mistral 4L centrifuge and was then stored at -20~'C for up to 2 weeks. Antibodies were purified by the method of Carter and Boyd (1979).

Immunological assays In the precipitin reaction, a known a m o u n t of purified IgG (10-100#g) was dissolved in 0 . 4 c m 3 of 0.1 M potassium phosphate buffer, pH 7.0. This was mixed with 10(~3000 ltg of homogenised guinea-pig liver or 5 ~ 5 0 0 / ~ g of homogenised epidermis (previously dialysed against 100 volumes of 0.1 M potassium phosphate buffer, pH 7.0) in 0.1 cm 3 of the same buffer, such that each of the samples contained antibody and a different, but known, a m o u n t of histidase activity. Samples were then incubated overnight at 4 C with controls lacking either antibody or enzyme. The resultant precipitates were removed by centrifugation in a Hettich maxi-fuge for 15min after which the supernatants were assayed for histidase activity. Double diffusion on agar gels was carried out by the method of Ouchterlony (1968). Purified antibody (100/lg) was placed in one well and histidase-containing samples in the other. Plates were incubated overnight in a saturated atmosphere at 37°C. The precipitin bands were visualised by washing the plates for 2 3 days with several changes of phosphate-buffer saline and staining with 0.04% Coomassic brilliant blue G250 dissolved in 3.5% perchloric acid for 1 hr, followed by destaining in 7% acetic acid.

lmmunoaffinity chromatography Sepharose 4B was activated by cyanogen bromide according to the method of Kumel et al. (1979). Purified antihistidase IgG was coupled to the activated Sepharose as follows: 10cm 3 of activated Sepharose was immediately mixed with 10 cm 3 of antibody (raised against purified guinea-pig hepatic histidase and purified by the method of Carter et al., 1979) dissolved in 0.1 M sodium carbonate buffer pH 8.5 (5 m g protein/cm3). Coupling was continued overnight after which the Sepharose was washed with 0.1 M glycine dissolved in the same buffer. The coupled Sepharose was then poured into 10cm 3 columns and equilibrated with 0.1 M Tris-HC1 buffer, pH 7.4.

525

Histidase in the guinea-pig Table 1. Purification of hepatic histidase TOTAL SAMPLE

VOLUME cm 3

TOTAL ACTIVITY n moles

TOTAL PROTEIN mg

urocanate/min

SPECIFIC ACTIVITY

PURIFICATION x

n moles urocanate/ m i n / m g protein

327.0

31,300

19,600

1,6

1.00

PARTICLE FREE SUPERNATANT

220.0

28,700

8,400

3.4

2.1

HEAT T R E A T M E N T AT 65°C FOR 3 MIN.

185.0

16,800

1,100

15.5

9.7

6.5

9,200

75.4

30 X 2.5cm D E A E ION E X C H A N G E COLUMN

27.0

4,900

9.5

70 x 1.5cm G200 SEPHADEX 'SUPERFINE' GEL FILTRATION COLUMN

4.5

920

20 x 2.5era QAE ION EXCHANGE COLUMN 0-0.7M NaCl GRADIENT

14.0

200

LIVER HOMOGENATE

AMMONIUM SULPHATE PRECIPITATION 45-50% S A T U R A T E D

RESULTS

Table 1 shows a typical purification of guinea-pig hepatic histidase. The enzyme was routinely purified over 1000-fold and polyacrylamide gel electrophoresis of the final preparation revealed a single band on gels run under both denaturing (SDS) and non-denaturing conditions. Comparison with molec-

122

77°0

526

331.0

0.68

1360

856.0

0.11

1860

1170.0

ular weight markers showed that the band on denaturing gels had a molecular weight of about 70,000 daltons. Analysis of the non-denaturing gels for histidase activity confirmed the single band as histidase. In addition the specific activity of histidase eluted from QAE Sephadex was constant for all the fractions which were subsequently pooled. This indicates that the hepatic histidase preparation obtained

Table 2. Purification of epidermal histidase TOTAL SAMPLE

VOLUME om 3

TOTAL ACTIVITY n moles

TOTAL PROTEIN mg

urocanate/min

100

5017

492.5

AMMONIUM SULPHATE PRECIPITATION 38-45% SATURATED

4

2234

10.1

40

664

6

10

70 x 1.5cm SEPHADEX G200 COLUMN z0 x 2.5om QAE SEPMADEX ION EXCHANGE C O L U M N 0-0.7M NaCI GRADIENT

PURIFICATION x

n moles urooanate/

min/mg protean

PARTICLE FREE SUPERNATANT O B T A I N E D FROM EPIDERMAL HOMOGENATE

30 x 2.5 cm DEAE SEPHADEX ION EXCHANGE COLUMN

SPECIFIC ACTIVITY

10.2

Io00

221

21.7

1.52

437

42.8

287

0.28

1025

100.5

79.8

0.06

1330

130.4

Combined epidermis from 20 pelts was normally used for purification.

526

ROGER L. ALLENet al.

by this procedure is a single homogeneous protein with one type of subunit, having an estimated molecular weight of 70,000 daltons. Table 2 shows the degree of purification for the epidermal enzyme. Polyacrylamide gel electrophoresis of the product revealed a single, though somewhat diffuse, protein band, on non-denaturing gels. Under denaturing conditions a single protein band with an estimated molecular weight of 70,000 daltons was always present, but 2 further, less distinct, protein bands with estimated molecular weights of 35,000 daltons and 100,000 daltons were also observed in some preparations. It seems likely that these bands are artefacts of the denaturation process. Both purified enzymes were found to have a broad pH optimum from pH 8.0 to pH 9.2. The Michaelis constants (Kin) of the two enzymes were determined at pH 7.0 (physiological pH) and at pH 9.2 (optimum pH) by Lineweaver Burk plots (Fig. 1). At pH 7.0 the Km values for the epidermal and hepatic enzymes were 0.360_+ 0.020mM and 1.000 _+ 0.013 mM respectively whereas at pH 9.2 the Km values were 0.340 + 0.028 mM and 0.553 + 0.022 mM respectively. (In each case the mean _+ SEM for 6 determinations using different preparations is quoted.) Thus the epidermal enzyme appears to have a similar affÉnity for its substrate at both pH

(a)

5040

301

V

2010

l/S mM

(bl

1

15

10 1

V 5

10 1/S m M

1'5

2'0

2'~

1

Fig. 1. Lineweaver Burk double reciprocal plots of epidermal and hepatic histidase. Purified epidermal and hepatic histidases were assayed as described in the Experimental section. The data presented are the means of 6 separate determinations for each enzyme. Figure l(a) shows the results at pH 7.0 and Fig. l(b) at pH 9.2; 0 - - 0 hepatic histase, • • epidermal histidase.

1,8

i

1 ~-~ 1.5

} ~ 1.0

1- o 0.5

0 --

~, 8 12 16 20 Reduced glutathione concentration (mM)

Fig. 2. The effect of reduced glutathione on hepatic and epidermal histidase activities. Purified hepatic and epidermal histidases were assayed in the presence and absence ol reduced glutathione as in Table 3, in either Tris HC1 buffer pH 9.2 or pH 7.4. 0 - - 0 hepatic histidase activity at pH 9.2; 0 - - 0 hepatic histidase activity at pH 7.4; A• epidermal histidase activity at pH 9.2; L~ z5 epidermal histidase activity at pH 7.4. The data are presented as means 4- SEM for 3 experiments. 7.0 and pH 9.2. whereas the hepatic enzyme has a significantly lower affinity at pH 7.0 than at pH 9.2. Low concentrations of reduced glutathione enhanced the enzymic activity of both hepatic and epidermal histidase preparations to a very similar extent at pH 9.2, but at pH 7.4 hepatic histidase activity was stimulated to a much greater degree than was epidermal histidase activity. Both enzymes are inhibited by higher concentrations of reduced glutathione at either pH 7.4 or pH 9.2. (Fig. 2). Similar effects were also observed with dithiothreitol. Ethylene diamine tetracetic acid (EDTA) was also shown to be inhibitory to both hepatic and epidermal enzymes. Table 3 indicates that hepatic histidase is more susceptible to EDTA inhibition than is the epidermal enzyme. Inhibition of both enzymes by high thiol concentrations or by EDTA could be overcome by incubating the enzyme with zinc ions (Table 4). On treatment with 10mM DTNB (5,5'-dithiobis-(2nitro)-benzoic acid) both epidermal and hepatic histidase preparations lost approximately 75% of their activity. Neither oxidized enzyme was affected by EDTA. However incubation of both oxidised enzymes with reduced glutathione or with dithiothreitol restored their original activity and susceptibility to inhibition by EDTA. Antibody raised against guinea-pig liver histidase was shown to precipitate histidase activity from either epidermal or hepatic preparations. Immunoprecipitation studies showed that antibody harvested after 5 weeks (1 booster injection) precipitated equal amounts of either hepatic or epidermal enzyme whereas antibody harvested after 10 weeks (3 booster injections) precipitated more hepatic enzyme than epidermal. Double diffusion studies on agar plates revealed complete identity between epidermal and hepatic histidase against antibody harvested after 5 weeks

Histidase in the guinea-pig

527

Table 3. The effect of EDTA on purifiedepidermaland hepatic histidases ENZYME

CONCENTRATION OF EDTA IN T H E A S S A Y S Y S T E M

ACTIVITY OF HISTIDASE RELATIVE TO THAT INCUBATED IN T H E A B S E N C E O F E D T A (,)

(~M) EPIDERMAL NISTIDASE

400

18 _+ 2.3

LIVER

"

400

4 + I. 2

EPIDERMAL

"

40

20 + 1.7

LIVER

"

40

7 +_ I, 2

EPIDERMAL

"

4

32 +_ 2.9

LIVER

4

15 + 1.7

EPIDERMAL

0.4

93 + 3.5

LIVER

0.4

82 +_ 2°9

0.04

98 + 2.3

0.04

98 + 2.9

EPIDERMAL LIVER

"

The results show the mean+ SEM for 3 experiments.

[Fig. 3(a)], but incomplete identity against antibody harvested after 10 weeks [Fig. 3(b)]. Immunoaffinity chromatography also revealed that the hepatic and epidermal enzymes had different affinities for antihepatic histidase on average 50-70% of bound hepatic histidase could be eluted with 3 M potassium thiocyanate whereas between 25 and 35% of bound epidermal enzyme could be so removed. Analysis of purified hepatic and epidermal histidase samples on Sepharose 6B showed that both enzymes were eluted at the same volume, coinciding

with a molecular weight of approximately 220,000 daltons. Under the same conditions, histidase activity from the supernatant of an epidermal homogenate eluted at the void volume of the column; histidase activity from the supernatant of a hepatic homogenate eluted partly at the void volume, but predominantly at the same elution volume as the purified enzymes. Reduction of these impure enzyme preparations with 10mM reduced glutathione prior to chromatography caused both hepatic and epidermal enzymes to elute at a volume corresponding to a

Table 4. The effect of Zn ions on the inhibitionof epidermaland hepatichistidaseactivitiesby dithiothreitoland EDTA ENZYME

pH

CONCENTRATION CONCENTRATION OF DITNIOO F E D T A IN T H R E I T O L IN ASSAY SYSTEM ASSAY SYSTEM (mMl (mM)

E P I D E R M A L 9.2 HISTIDASE 9,2

20

9.2

20

LIVER 9.2 HISTIDASE 9.2 9.2

CONCENTRATION OF Z I N C A C E T A T E IN ASSAY SYSTEM (mM)

-

20

A C T I V I T Y OF HISTIDASE RELATIVE TO THAT INCUBATED O N L Y IN THE PRESENCE OF HISTIDINE AND BUFFER

27% 0.4

120%

0.04

124%

20

-

0.5%

20

0.4

125%

20

0.04

118%

-

E P I D E R M A L 9.2 HISTIDASE " 9.2

0.04

23%

0.04

0.4

100%

9.2

0.04

0.04

100%

LIVER 9.2 HISTIDASE 9.2

0.04 0.04

0.04

100%

9.2

0.04

0.04

100%

5%

528

ROGER L. ALLENet al.

(a)

(b) Fig. 3. Double immunodiffusion analysis of hepatic and epidermal histidases. Double diffusion was carried out on agar plates as described in the Experimental section. (a) Top well: anti-hepatic histidase (100 ~g), harvested after 5 weeks. Right well: partially purified epidermal histidasc (100 ~tg). Left well: partially purified hepatic histidase (100l~g). (b) Top well: anti-hepatic histidase (100 pg), harvested after 10 weeks. Right well: partially purified epidermal histidasc (100k~g). Left well: partially purified hepatic histidase (100/tg).

molecular weight of 220,000 daltons. Treatment with DTNB had no effect on the elution pattern of either enzyme.

optimum pH (pH 9.2) were only marginal, whereas at physiological pH these differences were more apparent. Bhargava et al. (1976) claimed that rat epidermal and hepatic histidases had identical Km values, but these were measured only at pH 9.2. The effects of EDTA on the epidermal and hepatic enzymes were similar to those reported by Baden and Gavioli (1974) for the rat, with the hepatic enzyme being more susceptible to inhibition. Incubation of both enzymes with histidine prior to addition of E D T A delayed inactivation. This was also reported by Okamura et al. (1974), who suggested that the effect of EDTA was to chelate divalent metal cations involved at the active site, in which case the binding of histidine increases the acidity of the rat enzyme for its divalent cations. Both the inhibition by EDTA and by high thiol concentrations could be overcome by the addition of zinc ions. The differences in the effects of thiols on the two enzymes were again much more apparent at physiological pH than at the optimum pH of the enzymes. The initial immunological studies reported here suggested that the two purified enzymes were identical, but antibodies harvested after a prolonged exposure to antigen in rivo revealed that hepatic histidase appeared to contain at least one extra antigenic determinant. Kwapinski (1972) has shown that the production of antibody which reacts with minor antigenic determinants can indeed require prolonged exposure of the host animal to the antigen. The molecular weights of the two enzymes (220,000 daltons) and the proposed trimeric structure of three subunits each having a molecular weight of 70,000 daltons are in general agreement with the findings of Bhargava et al. (1976) for the rat enzymes, but are at variance with reports suggesting a structure of 6 subunits of molecular weight 35,000 daltons, also l\~r the rat enzyme (Brand and Harper, 1976). It is not clear whether the apparent polymerisation of the epidermal enzyme, compared to the monomerle lbrm of the hepatic enzyme is a genuine physiological difference or simply an artefact of the extraction procedure. Dhanam and Radhakrishnan (1974) have shown that histidase from monkey liver can reversibly polymerise and that this may be related to regulation of histidase activity. However the evidence presented here clearly indicates that the epidermal and hepatic histidases of the guinea-pig are not identical, either immunologically or kinetically. It appears likely that the two enzymes are products of identical genes but are posttranslationally modified in the epidermis or liver or both. Acknowledgement Financial support from S.E.R,C. and Unilever Research is gratefully acknowledged.

DISCUSSION The data presented here strongly favour the proposition that the epidermal and hepatic histidases of the guinea-pig arc similar but not identical enzymes. This conclusion supports the contention of Baden and Gavioli (1974), but is contrary to the view of Bhargava and Feigelson (1976), in both instances for the enzymes in the rat. Differences in K~ values for the two enzymes at

REFERENCES

Baden H. P. and Gavioli L. {1974) Histidase activity in rat liver and epidermis. J. Invest. Dermatol. 63, 479481. Baden H. P., Sviokla S., Mittler B. and Pathak M. A. 0968) Histidase activity in hyperplastic and neoplastic rat epidermis and liver. Cancer Res. 28, 1463 1468. Bhargava M. M. and Feigelson M. (1976) Studies on the mechanisms of histidase development in rat skin and

Histidase in the guinea-pig liver--I. Basis for tissue specific developmental changes in catalytic activity. Dev. Biol. 48, 212-225. Brand L. M. and Harper A. E. (1976) Histidine ammonia lyase from rat liver, purification, properties and inhibition by substrate analogues. Biochemistry 15, 1814-1821: Carter R. J. and Boyd N. D. (1979) Comparison of methods for obtaining high yields of pure immunoglobulin from severely hemolysed plasma. J. Immun. Meth. 26, 213-222. Dhanam M. and Radhakrishnan A. N. (1974) Purification and properties of histidine ammonia-lyase from monkey liver. Indian J. Biochem. Biophys. 77, 1-6. Feigelson M., Bhargava M. M. and Lamartiniere C. A. (1976) Progress in Differentiation Research (Edited by Muller-Berat N.), pp. 467-476, North-Holland, Amsterdam. Holbrook I. B. and Leaver A. G. (1978) Procedure to increase sensitivity of staining by Coomassie brilliant blue G.250 perchloric acid solution. Anal. Biochem. 75, 634-636. Kumel G., Daus H. and Mauch H. (1979) Improved method for the cyanogen bromide activation of agarose beads. J. Chromatogr. 172, 221-226. Kwapinski J. B. G. (1972) Methodology oflmmunochemical and Immunological Research, p. 254. Wiley Interscience, New York.

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Lamb R. A., Mahy B. W. J. and Choppin P. W. (1976) Synthesis of sendai virus polypeptides in infected cells. Virology 69, 116-131. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Mehler A. H. and Tabor H. (1953) Deamination of histidine to form urocanic acid in liver. J. Biol. Chem. 201, 775-784. Okamura H., Nishida T. and Nakagawa H. (1974) L-Histidine ammonia-lyase in rat liver--I. Purification and general characteristics. J. Biochem. 75, 139-152. Ouchterlony O. (1968) Handbook of Immunodiffusion and lmmunoelectrophoresis. Ann Arbor Sci. Publ., Ann Arbor. Schwartz E. (1961) Abbau yon Histidin zu Urocaninsaure in der Epidermis. Biochem. Zeitschrift 334, 415-424. Sedmak J. J. and Grossberg S. E. (1977) Rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G.250. Anal. Biochem. 79, 544-552. Zannoni V. G. and Ladu B. N. (1963) Determination of histidine ~-deaminase in human stratum corneum and its absence in histidinaemia. Biochem. J. 88, 160-163.