Comp. Biochem. Physiol., 1973, Vol. 44B, pp. 157 to 161. Pergamon Press. Printed in Great Britain

ISOELECTRIC VARIANTS OF MACACA IRUS SALIVARY ALPHA-AMYLASE

NILS JACOBSEN

Department of Microbiology, Dental Faculty, University of Oslo, Oslo, Norway

(Received 15 April 1972)

Abstract--1 . Isoelectric fractionation (LKB 8101 and 8102 columns, ampho- lines pH 3-10) of alpha-amylase produced by cultures of parotid and submandi- bular tissue from the Macaca irus monkey showed up to four amylase fractions, irrespective of different purification steps.

2. The pI of the fractions was reproducible, also after dialysis of alkaline fractions with EDTA solutions. The isoelectric variants of amylase, therefore, were probably not due to purification procedures or to charge differences caused by different content of divalent cations.

3. The M. irus and the human salivary amylase have proved to be very similar.

4. The "isozyme" pattern in the present report, as well as in most reports on human salivary amylase, shows a main fraction accompanied by several smaller ones.

5. This might indicate a non-genetic "ageing" process of the amylase molecules, as proposed for cytochrome c.

INTRODUCTION

DI~Em~NT electrophoretic procedures have revealed the presence of multiple forms of human salivary alpha-amylase (Millin & Smith, 1962; Muus & Vnenchak, 1964; N6rby, 1964; Lamberts et al., 1965; Masson et al., 1965; Wolf & Taylor, 1967; Hensten-Pettersen et al., 1970; Kauffman et al., 1970). Immunologic investigations indicated four amylase fractions in the parotid secretion, as compared to one in the submandibular secretion (Ellison, 1966). Isoelectric fractionation of parotid amylase from the Macaca irus monkey also indicated four components (Jacobsen, 1969).

Non-genetic "isozymes" may arise from the purification procedure, or may be based on charge differences only (review by Leese, 1969). Isoelectric splitting of a protein may also be caused by the difference in the content of calcium (Gladhaug & Prydz, 1970).

Human salivary amylase contains calcium necessary to stabilize the molecules (Hsiu et al., 1964; Stein et al., 1964), whereas a dimer form of Bacillus subtilis amylase is stabilized by Zn ~+ ions (Kakiuchi et al., 1964).

157

158 NILS JACOBSEN

T h e present exper iments were carried out to s tudy the pat tern of isoelectric variants of M. irus parot id a lpha-amylase after different purification steps and after removal of divalent cations. I t was also of interest to determine whether variant forms of submand ibu la r amylase occur red in this m o n k e y species.

M A T E R I A L S AND M E T H O D S

Treatment of amylase samples Samples of parotid and submandibular alpha-amylase from Macaca irus were obtained

from in vitro cultures of glandular tissue in a synthetic culture medium, as described pre- viously (Jacobsen, 1969). Medium samples, containing the in vitro secreted amylase, were subject to different treatment before electrofocusing: (1) Gel filtration on Sephadex ® G-100 columns in 0'05 M phosphate buffer, pH 7'0, at room temperature. (2) Dialysis against distilled water at 4°C, centrifugation for 10 min at 1100 g, sediment discarded. (3) Dialysis against 0"85 % NaCI solution at 4°C, centrifugation for 10 min at 1100 g, sediment discarded (negligible). (4) Purification by a combined alcohol/glycogen precipitation technique with final lyophilization, as described by Loyter & Schramm (1962). The samples were dissolved in 0"05 M phosphate buffer, pH 7"0.

Electrofocusing Fraetionation of the samples was carried out in a pH/sucrose gradient, using LKB 8101

and 8102 columns with inner and outer cooling. Ampholines in the pH range 3-10 were used (LKB 8141), following the procedure recommended by the manufacturers. After 48 hr, fractions of 3 or 10 ml, for small and large eolumns respectively, were eollected, and the pH determined.

Chelation and refocusing of the alkaline fractions An alkaline fraction of parotid amylase (pI 8"25-8"71 from monkey No. 4, Table 1) was

lyophilized and dissolved in phosphate buffer, 0"05 M, pH 6"8. Parts of this solution were dialyzed against 100 vol. of 0"05 M EDTA, pH 6"8, for 62 hr, according to Stein et al. (1964). The alpha-amylase was determined before and after E D T A dialysis, and the dialyzed material was refocused on the LKB 8101 eolumn with the pH 3-10 ampholines. Un- treated solution of amylase from the same fraction was also refocused (control).

Another alkaline fraction (pI 7"1, from monkey No. 5, Table 1) was dialyzed against the E D T A solution for 72 hr. The Ca-EDTA complex was removed by dialysis for 24 hr against distilled water, after which parts of the fraction were refocused and parts dialyzed against a solution of CaC12 for 2 days. A parallel sample from the same pI fraction was dialyzed against distilled water, eontaining CaCI~, and refocused (control).

The alpha-amylase activity (Jacobsen & Hensten-Pettersen, 1970) was followed during the procedure, and the pI determined after refocusing.

RESULTS AND DISCUSSION Isoelectric fractionation of parotid amylase from the M. irus monkey gave from

two to four peaks of amylase activity at isoelectric points about 5-5, 6.0, 7-3 and 8.2 (Table 1). The components occurred irrespective of mild purification steps (dialysis and gel filtration) or more severe purification procedures (glycogen alcohol precipitation and lyophilization). The components at pI's of 6.0 and 8.2 were most consistent.

Removal of divalent cations from alkaline fractions of parotid amylase by chelation did not lead to reduced pl values (Table 3), but the amylase activity was

ISOELECTRIC VARIANTS OF M.4C.4C.4 1RUS SALIVARY ALPHA-AMYLASE 159

TABLE 1--ELEcTROPOCUSING OF ALPHA-AMYLASE PRODUCED BY in vitro CULTURES OF PAROTID

GLAND TISSUE FROM m . iry.$. L K B COLUMNS 8101 AND 8102

Treatment No. of Isoelectric Percentage Percentage Monkey of amylase points amylase recovery

No. sample peaks of peaks in peaks of amylase

1 Gel-filtrated 4 5-16-5"32 25 6"08 25 7"38 7 8-38 43

2 Dialyzed against 4 5.18 16 distilled water 5.64-5.94 37

7-30 14 8"20 33

3 Dialyzed against 3 4 5.55 8 distilled water 6"08 62

7.38 7 8.25-8-71 22

4 Alcohol/glycogen 3-4 5.55 14 precipitation 6"15 45

6"65 20 7.18 20

5 Alcohol/glycogen 2 6.22 75 * precipitation 7" 10

83

97

60

* The residual amylase was eluted in the pH range 4"5-8"5 without distinct peaks.

TABLE 2~ELECTROFOCUSING OF ALPHA-AMYLASE PRODUCED BY in vitro CULTURES OF

SUBMANDIBULAR OLAND TISSUE FROM M. irus. LKB COLUMNS 8101 AND 8102

Treatment No. of Isoelectric Percentage Percentage Monkey of of points amylase recovery

No. sample amylase peaks of peaks in peaks of amylase

2 Dialyzed against 1 5.81-6.31 100 97 0"85% NaCI

3 Dialyzed against 3 5.18-5.74 6 60 distilled water 7-22 14

8"18 80

reduced to about 30 per cent. T h e addition of CaCla did not restore the activity. Whether a complete denaturation of the enzyme by E D T A would lead to a reduced isoelectric point was not investigated.

T h e first submandibular amylase samples showed one fairly broad peak at a p I of about 6.0. T h e second sample showed peaks at p I ' s of 5.5, 7.3 and 8.2, of which the last fraction contained 80 per cent of the total activity (Table 2).

160 NILS JACOBSEN

TABLE 3 - - I s O E L E C T R I C POINTS OF ALKALINE FRACTIONS OF PAROTID ALPHA-AMYLASE,

REFOCUSED BEFORE AND AFTER E D T A TREATMENT

Refocusing First Refocusing after EDTA

Sample focusing (control) treatment

1 8"25-8.71 8"50 8-58 2 7.10 6"12-7"13 6"39-7'08

The origin of the isoelectric variants of M . irus parotid amylase could not be explained by the purification procedure, or by a difference in the content of divalent cations. Nor were the "isozymes" found only in the parotid secretion, as suggested by Ellison (1966) for the human enzyme. The results more likely indicated two main types of salivary amylase in both glands, one acidic and one alkaline. Polymorphism in the monkey population was ruled out since all samples were taken from single specimens.

The present isoelectric variants of monkey salivary amylase are probably parallel to the electrophoretic fractions found in the human salivary amylase. A close similarity between these two enzymes has been reported concerning molecular weight and amino acid composition (Jacobsen & S6nju, 1971). Whether these amylase variants are genetically determined, such as lactic dehydrogenase (Kaplan, 1963), is still uncertain. Most observations to this date are based on charge differences only.

A common observation in the present, as well as in previous reports, is one main fraction dominating the "isoamylase" picture. This pattern might indicate an "ageing" process of the amylase molecules similar to the one suggested for multiple forms of rat cytochrome c (Flatmark & Sletten, 1968). In this process, the deamidation of asparagine and glutamine residues has been proposed as an explanation of the structural changes (Robinson et al., 1970).

Preparative use of the electrofocusing technique may prove to be a sensible approach to further analyses of multiple forms of salivary amylases.

REFERENCES ELLmON S. A. (1966) Salivary antigens. J. dent. Res. 45, 644-654. FLATMARK T. & SLETTEN K. (1968) Multiple forms of cytochrome c in the rat. Precursor-

product relationship between the main component Cy I and the minor components Cy II and Cy III in vivo. J. biol. Chem. 243, 1623-1629.

GLADHAUG h. & PRYDZ H. (1970) Purification of the coagulation factors VII and X from human serum. Some properties of factor VII. Biochim. biophys. Acta 215, 105-111.

HN~ISTEN-Pm~rERSEN A., $61'~1u T. & DE BEST T. (1970) Human salivary alpha-amylase. Puri- fication and amino acid composition. J. dent. Res. 78, 440-446.

HsIu J., FISCHER E. H. & STEIN E. A. (1964) Alpha-amylase as calcium-metalloenzymes--I I. Calcium and the catalytic activity. Biochemistry 3, 61-66.

JAeOBS~'~ N. (1969) In vitro production of alpha-amylase by monkey parotid gland. Arch. oral Biol. 14, 679-683.

ISOELECTRIC VARIANTS OF M A C A C A I R U S SALIVARY ALPHA-AMYLASE 161

JACOBSEN N. & HENSTEN-PETTERSEN A. (1970) Salivary amylase---I. An assay method of alpha-amylase. Caries Res. 4, 193-199.

JACOBSEN N. & SSNJu T. (1971) Molecular weight and amino acid composition ofMacaca irus parotid alpha-amylase. Scand. ~7. dent. Res. 79, 193-201.

KAKIUCHI K., KATO S., IMANISHI A. & ISEMURA T. (1964) Association and dissociation of Bacillus subtilis s-amylase molecule--II. Studies on monomer-dimer transformation by gel filtration, ft. Biochem. 55, 102-109.

KAPLAN N. O. (1963) Symposium on multiple forms of enzymes and control mechanisms--I. Multiple forms of enzymes. Bact. Rev. 27, 155-169.

KAOVlCMAN D. L., ZAOER N. I., COHEN E. & KELLER P. J. (1970) The isoenzymes of human parotid amylase. Arch. Biochem. 137, 325-339.

LAMBERTS B. L., MEYER T. S. & LOSEE F. L. (1965) Isolation of amylolytic fractions from human parotid saliva. Fedn Proc. Fedn Am. Socs exp. Biol. 24, 441.

LEESE C. L. (1969) Enzymes and isoenzymes. In Homeostatic Regulators. (Edited by WOLSTENHOLME G. E. W. & KNIGHT J.), pp. 144-164. Ciba Foundation Symposium. Churchill, London.

LOYTER A. & SCHRAMM i . (1962) The glycogen-amylase complex as a means of obtaining highly purified s-amylases. Biochim. biophys. Acta 65, 200-206.

MASSON P. L., CARBONARA A. O. & HEREMANS J. F. (1965) Studies on the proteins of human saliva. Biochim. biophys. Acta 107, 485-500.

MILLIN D. J. & SMITH M. H. (1962) Gel filtration and chromatography of human salivary proteins. Biochim. biophys. Acta 62, 450-455.

MUU8 J. & VNFaNCHAK J. M. (1964) Isozymes of salivary amylase. Nature, Lond. 204, 283- 285.

NORBY S. (1964) Electrophoretic non-identity of human salivary and pancreatic amylases. Expl Cell Res. 36, 663-666.

ROBINSON A. B., McK~Row J. H. & CARY P. (1970) Controlled deamidation of peptides and proteins: an experimental hazard and a possible biological timer. Proc. hath. Acad. Sci. U.S.A. 66, 753-757.

STEIN E. A., HsIu J. & FISCHER E. H. (1964) Alpha-amylase as ealeium-metalloenzymes--I. Preparation of calcium-free apoamylases by chelation and electrodialysis. Biochemistry 3, 56-61.

WOLV R. O. & TABOR L. L. (1967) Isoamylases of human parotid saliva. Nature, Lond. 213, 1128-1129.

Key Word Index--Salivary amylase; isoelectric fractionation; Macaca irus; ageing of protein; alpha-amylase.