Initial sites of insulin cleavage and stereospecificity of carboxyl proteinases from Aspergillus sojae and Pycnoporus coccineus

Biochimica et Biophysica Acta, 700 (1982) 247-253

247

Elsevier Biomedical Press BBA 31042

INITIAL SITES OF INSULIN CLEAVAGE AND STEREOSPECIFICITY OF CARBOXYL * PROTEINASES FROM ASPERGILLUS S O J A E AND P Y C N O P O R U S COCCINEUS EIJI ICHISHIMA a.**, MAKOTO EMI a, EIJI MAJIMA a, YASUHIRO MAYUMI a HIROYUKI KUMAGAI a, KAZUYA HAYASHI b and KATSUMI TOMODA c

Laboratory of Enzymology and Microbial Chemistry, Tokyo NSk6 University, Fuchu, Tokyo 183, b Central Research Laboratories of Kikkoman Corp., Nodg, Chiba Prefecture 278, and c Research Laboratories of Fermentation Products, Takeda Chemical Industries, Ltd., Osaka 532 (Japan) (Received August 31st, 1981)

Key words: Insulin cleavage; Carboxyl proteinase; Stereospecificity; Proteinase

Initial cleavage sites of native insulin at a pH of about 3 and stereospecificity were investigated by fungal carboxyl proteinases (EC 3.4.23.6) from Aspe~fillus sojae, a species of fungi impedecti, and Pycnopurns coccineus (formerly designated Trmnetes sangu/nea), a wood deteriorating Basidiomycete, respectively. Fungal carboxyl proteinases were used as a model of vertebrate insulin degradation. A. sojae carboxyl proteinase I primarily hydrolyzed two peptide bonds located on the surface of native insulin monomer, the B16-B17 (Tyr-Leu) and B24.B25 (Phe-Phe) bonds, and secondarily the buried bonds, A15-A16 (Gln-Leu), BIS-B16 (Leu-Tyr) and B14-B15 (Ala-Leu), at pH 3.2 and 30°C. The initial cleavage sites of A. sojae carboxyl proteinases I towards native insulin were not identical with the initial cleavage sites towards the oxidized B chain of insulin. P. cocc/neus carboxyl proteinase I I selectively hydrolyzed B14-BI5 (Ala-Leu), BI6-BI7 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds in the native insulin at pH 2.7. Based on these findings we suggest that the stereospecificity of the fungal carboxyl proteinases is similar to that of cathepsin D (EC 3.4.23.5), and that the synthesis and degradation of insulin may occur in microorganisms.

The proteolytic degradation of insulin is an integral part of its interaction with the cell, possibly playing a role in insulin action [1]. Identification and characterization of intermediate degradation products thus become of importance. There are two processes by which insulin can be degraded. The disulfide bonds can be reduced by enzyme glutathione-insulin transhydrogenase (EC 1.8.4.2), resulting in the production of A and B chain. The peptides are then susceptible to further degradation by nonspecific cellular hydrolases. The * These enzymes are now more correctly termed 'aspartic' according to the Enzyme Nomenclature, Recommendations 1978, Supplement 2, (Eur. J. Biochem. 116, 423-435 (1981)). ** To whom correspondence should be addressed. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

other insulin-degradation process is the direct proteolytic degradation of the molecule by the enzyme, insulin protease [2]. Duckworth et al. [3] showed that the digest of insulin protease was composed of an intact A chain and a B chain cleaved between residue B16 and B17, with three peptide chains held together by disulfide bonds. Recently Duckworth and Halban [1] reported that three peptides, B1-B10, B1-B16 and B1-B25 or 26, were identified from the hydrolyzate of semisynthetic [3H]Phem-insulin with purified insulin protease for various times. Thus, exposure of insulin to the proteolytie insulin degrading enzyme results in products of similar molecular weight to native insulin but with one or more cleavages in the B-chain.

248

In earlier papers from this laboratory, the peptide bond specificities of carboxyl (acid) proteinases were investigated. Primary cleavage sites at the Phe24-Phe 25 and LeulS-Tyr 16 in the oxidized B chain of insulin with Aspergillus sojae carboxyl proteinase I (EC 3.4.23.6) [4] were identical with those of human erythrocyte cathepsinD (EC 3.4.23.5) [5]. The primary cleavage site at Phe 24Phe 25 in the oxidized B chain of insulin with Pycnoporus coccineus carboxyl proteinase I a (EC 3.4.23.6) [6,7] was identical with that reported in the work on human erythrocyte cathepsin D [5]. The purposes of the study are as follows: (a) understanding of the mechanism of insulin degradation by carboxyl proteinases in eukaryotic microorganisms; (b) comparison with insulin degradation by proteinases in vertebrates: cathepsin D, insulin-specific protease and others; (c) to learn whether the presence of insulindegrading enzymes in microorganisms supports the hypothesis that insulin biosynthesizes in eukaryotic microorganisms [8]. Materials and Methods

Materials. Crystalline bovine insulin (lot 57590) was purchased from Fluka AG, Buchs, Switzerland. L-Leucyl-L-tyrosine (lot 116C-0297) was purchased from Sigma Chemical Co. L-Tyrosyl-Lleucine was purchased from the Protein Research goundation, Osaka. Crystalline Penicillum janthinellum acid carboxypeptidase was prepared according to Ref. 9. The enzyme is now commercially available from the Protein Research Foundation, Osaka (Code 3502). Carboxyl proteinases. Purification of A. sojae carboxyl proteinase I was performed by the method described previously [4]. Purification of P. coccineus ( Trametes sanguinea) carboxyl proteinase I s was also performed by the previously described method [6]. Separation and identification of peptide from the digest of native insulin. Native insulin (35 mg A. sojae proteinase and 23 mg P. coccineus proteinase) was dissolved in 35 ml of 0.01 M sodium acetate buffer, pH 3.2, 6.55 nkatal (e/s; 1:1630, mol/mol) of A. sojae carboxyl proteinaseI or 6nkatal (e/s; 1:4862, mol/mol) P. coccineus carboxyl proteinase I s was added to the solution;

the pH was then adjusted to 3.2 for A. sojae proteinase and 2.7 for P. coccineus proteinase with dilute HC1. The mixture was incubated at 30°C for 50 rrfln A. sojae proteinase and 45 min for P. coccineus proteinase. One drop of concentrated NH4OH was added to inactivate the enzyme. The samples of hydrolysates were stored at - 2 0 ° C , and then the frozen digest was lyophilized. The freeze-dried digest of native insulin was separated with or without performic acid oxidation. First, the peptides in the freeze-dried digest of native insulin were separated directly on paper electrophoresis at pH 6.5 for 180 min and then by paper chromatography according to the previously described method [4,6,10]. The use of abbreviations for the peptides, such as An-l, etc., and Pn-l, etc., denotes peptides obtained with A. sojae proteinase and P. coccineus proteinase, respectively. The letters a, n and b refer to acidic, neutral or basic peptides, respectively. The peptide An-2, as well as An-1 and Ab-1, was separated on gel filtration with a Sephadex G-25 column (2 × 74 cm) using 1 M acetic acid. Eluates of the fraction from 144 to 146 ml were collected and examined. Pn-5 isolated from the fingerprint map was further purified by gel filtration a Sephadex G-50 column (2 × 70 cm) using 1 M acetic acid. Eluates of the fraction from 142 to 152 ml were collected and examined. Second, the freeze-dried digest of native insulin was pooled and the disulfide bonds broken with performic acid as described by Craig et al. [11]. The freeze-dried digest of native insulin was dissolved in 1 ml ice-cold formic acid and 2 ml ice-cold performic acid were added. After 2 h at 0°C 30 ml ice-cold water were added to the mixture to stop the reaction. The oxidized peptides in the reaction mixture were lyophilized and 10 mg of the peptide were then dissolved in 100 #1 of 1 M NH4OH and separated on paper electrophoresis and by paper chromatography according to the previously described method [4,6,10]. Abbreviations of the oxidized peptides, such as Ano-1, etc., and Pno-1, etc., show the oxidized peptide obtained with A. sojae proteinase and P. coccineus proteinase, respectively. Amino acid analysis. Freeze-dried peptides were dissolved in 2ml 5.7 M HC1 containing 5 #1 2mercaptoethanol and 2 drops of 5% phenol, and

249

were hydrolyzed at I10°C for 24h. Hydrolysates of peptides were examined with a Hitachi amino acid analyzer, Model 834-30. The recovery (/~mol) of peptide was calculated from the data of amino acid composition. Determination of the N-terminal amino acid of peptides. The N-terminal amino acids of the peptides were determined by the DNP-method of Sanger [12]. The DNP-amino acid was identified by paper chromatography with the solvent system 1 M NaHzPO4/0.5 M Na2HPO 4 buffer, pH 6.0, and tert- amylalcohol/potassium biphthalate buffer, pH 6.0. Furthermore, the DNP-amino acid obtained was heated for 8 h in a sealed tube with 28% NH4OH [13] and then the amino acid was identified by a Hitachi amino acid analyzer. Determination of C-terminal amino acid of

peptides. Release of the C-terminal amino acids from the peptides was performed by Penicillium janthineilum acid carboxypeptidase [14-16] at pH 3.7 and 30°C.

Determination of Leu-Tyr. The dipeptide, Leu-Tyr, which is a peptide corresponding to Pn-1 and Pno-1, was identified directly by a Hitachi amino acid analyzer. Results

Cleavage sites of native insulin with Aspergillus sojae carboxyl proteinase I Two peptides, An-1 and Ab-1, were obtained by paper electrophoresis and paper chromatography from the 50-min digest of native insulin with A. sojae carboxyl proteinase I at pH 3.2. The An-2

TABLE I AMINO ACID COMPOSITIONS OF PEPTIDES OBTAINED FROM THE 50-MIN DIGESTS OF NATIVE INSULIN BY A S P E R G I L L US SOJA E CARBOXYL PROTEINASE I AND P Y C N O P O R US C O C C I N E U S CARBOXYL PROTEINASE I a Amino acid

Peptides (residues/molecule) Obtained by A. sojae carboxyl proteinase I

CySO 3H Asp Thr Ser Glu Pro Gly Ala Cys Val Ile Leu Tyr

Obtained by P. coccineus carboxyi proteinase I a

An-I

An-2

1,85 (2)

2.25 (I) a

2,05 (2)

2.78 (3) 5.22 (5)

Pn-I

Pn-5

Pb-1

0.51 2.96 (3)

I.OO(1)

0.97 (1) 3.04 (3) 7.00 (7)

0.88 (I)

1.08 (I) 2,12 (2) nd 1,28 (I)

2,55 (2) 1.52 (2) nd 2.99 (4) a 1.12 (1) 4.00 (4) 1.70 (2) 1.15 (I)

Phe

2.00 (2) 1.08 (1) 0.98 (I)

Lys His Arg Recovery (#mol) N-terminal C-terminal Suggested segment

1.00 (I) 0.034 Leu Phe Ai6_.,._ A21

0.079 Gly, Phe Gin, Tyr AI ._~AI5

$

$

BI7_.2._sB24

BI s, BI6

a

Ab-1

1.03 (1)

1.20 (I) 1.16 (1) 1.00 (1)

0.65 (1) 1.oo (1)

2.79 (4) 1.69 (2) nd 5.00 (5) 0.94 (1) 5.18 (6)

2.38 (3)

0.49 (I) 0.74 (I) 1.02 (I)

1.83 (2)

1.71 (2) 0.085 Phe

0,102 Leu

2.17 (2) 0.49 (1) 0.079 Gly, Phe, Leu

B25-B30

BI5-B16

AI ,

Uncertain because of the relatively high background, nd, not determined.

1.06 (1)

0.085 Phe .A21

BI - B I 4 B I 7 - B24

B25-B30

250 TABLE II RELEASE OF C-TERMINAL AMINO ACID RESIDUES FROM An-2, Ano-2, Ano-3, Pno-2 AND Pno-3 PEPTIDES BY PENICILLIUMJANTHINELLUM ACID CARBOXYPEPTIDASE AT pH 3.7 AND 30°C Peptide

Incubation

9 27

Concentration (nmol)

An-2 Ano-2 Ano-3

146 146 200 70

10 50 10 30

0.220 0.220 0.220 0.220

Pno-2

50

20

0.345 0.345 0.345 0.345

a

70

48 5 15

min

Amino acid released (nmol)

Name

Pno-3

h

Acid carboxypeptidase (nkatal)

Gln

His

Leu

8 8

3 4

Val

Glu

Ala

Leu

Tyr

4 18

17 42 8 29

16 42 17

15 33 55

7

9 31 tr 8

3 15 43 43

2 18 43 43

13 23 57 50

4a 8a

13 12

Uncertain because of the relatively high background, tr, trace.

peptide fragment was obtained by the gel filtration procedure with Sephadex G-25. Amino acid compositions of the peptides obtained are shown in Table I. Rdease of the C-terminal amino acids from peptides An-1 and An-2 were performed with P. janthinellum acid carboxypeptidase [14-16] at pH 3.7, as shown in Table II. The results of the determination of the N-terminal amino acid of the peptides are summarized in Table I and Fig. 1A. The peptide Ab-1 was identified as B25-B30. An-1 was identified as the two peptides AI6-A21 and B17-B24 with interchain disulfide at A20 connected to the B chain at 19. The peptide An-2 was identified as peptides A1-A15 and B1-B16 with interchain disulfide at A7 connected to B7. The results are summarized in Fig. 1A. Ten oxidized peptides were obtained after performic acid oxidation from the 50-rain digest of native insulin. The results are shown in Fig. lB. The fingerprint map including Aao-1, Aao-2, An 0 -2 and Ab0-1 obtained from the oxidized hydrolysates of native insulin with A. sojae carboxyl proteinase I was identical with that including A-1, A-2, N-3 and B-1 obtained from the hydrolysates of the oxidized B chain of insulin [4]. Furthermore, acidic peptides Aao-3, Aao-4 and Aao-5 were obtained from the fingerprint map. The Nterminals of peptides Aao-1, Aao-2 and Abo-1 were identified as tyrosine, leucine and phenylalanine, respectively. Release of the C-terminal amino acids from peptides Ano-2 and Ano-3 was

•, sI I ~ol I ,s $ 2o I~IVEOCCASVCS,VOI ILENYC NI BI

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0.13 0.25 A1 5~ 10 15'~ 20 ~G IV E QIC°C'A $ V C S L Y O . L E N YC'N: i,(

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i l<

An,-3(0.016)

i(

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Aa.-1 (0.034) i i ,< /~-2(0.O75) '('-:+:~i ÷ 'is'!

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V EA:L=Y:L VC"GE RG F : F Y T P K A ; 0.08

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Fig. 1. Summary of the stereospecificityof carboxyl proteinase I from Aspergillus sojae towards native insulin at pH 3.2 and 30°C. A. The enzymic digest of native insulin was directly separated as described in the text. B. The enzymic digest of native insulin was oxidized with performic acid, and then the oxidized digest was separated. Abbreviations of amino acids are in the alphabetical system. C* indicates cysteine suifonic acid. The values in parentheses denote the uncorrected recovery (/.tmol) of pepdde. The perpendicular arrows indicate the bond split and the relative rate of hydrolysis at the B24-B25 bond. The horizontal arrows indicate residues removed by the following procedures: - , , identified by the DNP-method; ,--, amino acid released with Penicillium janthinellum acid carboxypeptidase [13-15], the degree of hydrolysis being as follows: . . . . .

251

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30

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Pn.-4(0.115 )

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t

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Fig. 2. Summaryof the stereospecificityof Pycnoporuscoccineus carboxyl proteinase I a towards native insulin at pH 2.7 and 30°C. A. The enzymicdigest of native insulin was directly separated as described in the text. B. The enzymicdigest of native insulin was oxidizedwith performic acid, and then the oxidized digest was separated by the methoddescribed above. Symbolsare the same as thoseof Fig. 1. performed with P. janthinellum acid carboxypeptidase [14-16] at pH 3.7 and 30°C as shown in Table II. The C-terminal amino acids of Ano-2 and Ano-3 were identified as tyrosine and alanine, respectively. The results of the present experiment indicate that A, sojae carboxyl proteinase I primarily hydrolyzed two peptide bonds, the B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds, and secondarily the AI5-A16 (Gln-Leu) bond, at the early stage of 50-rain incubation at pH 3.2 and 30°C. Furthermore, minor cleavage sites at BI5-B16 (Leu-Tyr), B14-B15 (Ala-Leu) and A5-A6 (Gln-Cys) were observed.

Cleavage sites of native insulin with Pycnoporus coccineus carboxyl proteinase I~ Three peptides, Pn-1, Pn-5 and Pb-1, were obtained by paper electrophoresis and paper chromatography from the 45-min digest of native insulin by P. occineus carboxyl proteinase at pH 2.7. Amino acid compositions of the peptides obtained are shown in Table I. The peptide Pn-1 was identified as Leu-Tyr by two different methods, the usual method and the direct method. The retention time of direct determination of Leu-Tyr with an amino acid analyzer was 42-43 min; that of TyrLeu was 44-45 rain. The N-terminal amino acids of Pn-1 and Pb-1 were identified as leucine and

phenylalanine, respectively. The peptide Pb-1 was identified as B25-B30. The Pn-5 peptide has three N-terminals, glycine, phenylalanine and leucine. The molar ratio of N-terminal amino acids glycine/phenylalanine/leucine is 1.0: 1.2 : 0.3. The third N-terminal amino acid, leucine, is a new one after enzymic hydrolysis with P. coccineus carboxyl proteinase I a. The hydrolyzed product Pn-5 seems to have three peptides, A1-A21, B1-B14 and B17-B24, with two interchain disulfides at A7 and A20 connected to the B chain at B7 and B19. Summarized results are shown in Fig. 2A. Seven oxidized peptides were obtained after a performic acid oxidation procedure from the 50rain digest of native insulin. The fingerprint map including Pbo-1, Pno-1, Pno-2 , Pno-3 and Pao-3 peptides obtained from the oxidized hydrolysates of native insulin with P. coccineus carboxyl proteinase I a is identical with that including B-l, N-l, N-2, N-3 and A-3 obtained from the hydrolysates of the oxidized B chain of insulin described in the previous paper [6]. Furthermore, acidic peptides Pao-4 a, Pao-4 b and Pao-4 c were obtained on the fingerprint map. Amino acid compositions of the three peptides were identical with that of the oxidized A chain of insulin. The N-terminals of Pno-1, Pao-3 and Pbo-1 peptides were identified as leucine, leucine and phenylalanine, respectively. Release of the Cterminal amino acids from the peptides Pno-2 and Pno-3 was performed with P. janthinellum acid carboxypeptidase [14-16] at pH 3.7 and 30°C, as shown in Table II. The C-terminals of the Pno-2 and Pn o-3 peptides were tyrosine (B 16) and alanine (B14), respectively. The results of the present experiment showed that P. coccineus carboxyl proteinase I a primarily hydrolyzed three peptide bonds in the B chain of native insulin, the B16-B17 (Tyr-Leu), B24-B25 (Phe-Phe) and B14-B15 (Ala-Leu) bonds at pH 2.7 and 30°C. P. coccineus carboxyl proteinase Ia did not hydrolyze the A chain of native insulin molecules at the early stage of 45-rain incubation at pH 2.7. Discussion

Molecular mechanism and stereospecificity of microbial proteinase degradation of insulin

252

C. ~) 3-fold axis B 30

(. ~ 3-fold axis

s

B~

q

B

B

NHa

N~

2-fold a×is

2-fold axis ~

~ ~ ahelixinBchain

A :o':L

~

s

~_.s~

----:d

rOK

A : .eoc ~c~ ~

~'~'l t~f/ "~-| ahelixinBehain

OH

1

2

Fig. 3. Stereographic views of the initial cleavage sites of Aspergillus sojae carboxyl proteinase I and Pycnoporus co~z'ineus carboxyl proteinase I a towards native insulin. The wide arrows indicate the bond split. 1, Aspergillus soja carboxyl proteinase I. 2, P.vcnoporus coccineus carboxyl proteinase I a.

The zinc-binding hexamer in crystalline insulin corresponds to the 36000 molecular weight species present in solution at neutral pH in the presence of zinc ions, and is composed of three dimers each of 12000 molecular weight [17,18]. Doty et al. [19] have shown that below pH 2.2 the monomer-dimer equilibrium (12 ,~ 21) can be isolated with a dissociation constant of 0.0625 • 10 -5. When Frederiq and Neurath [20] submitted insulin to sedimentation and diffusion experiments carried out in the presence of dihydrogen phosphate at pH 2.6, the resulting data yielded a minimum molecular weight value of approx. 6000. According to this evidence, the molecular structure of insulin used in this experiment at pH 2.7 or 3.2 may be partly dissociated as a monomer with a value of 6000. Major cleavage sites in native insulin molecules with A. sojae carboxyl proteinase I and P. coccineus carboxyl proteinase I a are shown in Fig. 3. Cleavage site B24-B25 lies in the fl-plated structure of the insulin dimer, and cleavage site B16-B17 lies in the a-helix conformation. Previous papers [4,6,7,10] showed that the Sj-P~ interactions (as

defined by Schechter and Berger [21]) might be important in increasing enzyme-substrate affinity and turnover rate for fungal carboxyl proteinases. In the insulin monomer the side chains of P~ position at B16-B17 and B24-B25 bonds are on the surface while the side chains of A16 and B15 leucines are buried [18]. Present results suggest that the insulin cleavage site in the native insulin monomer may be B24-B25 and B16-B17 bonds by the two fungal carboxyl proteinases, A. sojae carboxyl proteinaseI and P. coccineus carboxyl proteinase I a. The second cleavage site may be A15-A16 bond for A. sojae carboxyl proteinaseI and B14-B15 bond for P. coccineus carboxyl proteinase I a. The stereospecificity and initial cleavage sites of A. sojae carboxyl proteinase I towards native insulin at pH 3.2 were not identical with the peptide bond specificity and initial cleavage sites towards the oxidized B chain of insulin, while the initial cleavage sites of P. coccineus carboxyl proteinase Ia towards native insulin were identical with those towards the oxidized B chain of insulin.

253

Comparison with results obtained with proteases from vertebrates Duckworth et al. [3], have reported that the initial cleavage of insulin at pH 7.5 by insulin protease is between residues 16 and 17 (Tyr 16-1eu17) in the B chain, resulting in a molecule consisting of three peptide chains held together by disulfide bonds. Recently Duckworth and Halban [1] confirmed that the initial cleavage sites in the native insulin may be B10-BII, B16-B17 and B25-B26 or B26-B27 bonds by insulin protease. Like human erythrocyte cathepsin D [5], A. sojae carboxyl proteinase [4], A. saitoi carboxyl proteinase [10] and P. coccineus carboxyl proteinase I a [7], porcine pepsin C [22] have high affinity for the Phe24-Phe 25 bond in the oxidized B chain of insulin. Hypothesis that insulin biosynthesized and degraded in fungi The polypeptide hormone insulin is required for normal glucose homeostasis in vertebrates. Insulin deprivation results in diabetes, a disease affecting up to 5% of the human population in the United States [23]. In some animals there are two insulin genes; however, in most others, including humans, a single gene is present. Recently Owerbach et al. [24] found that the insulin gene is located on chromosome 11 in humans. Roith et al. [8] also recently found immunologically and biologically active insulin in unicellular eukaryotes (Tetrahymena pyriformis, Neurospora crassa, and Aspergillus fumigates) grown in defined media in the absence of macromoelcules. The findings suggest that insulin did not arise evolutionarily in the intestinal or neural tissues of primitive vertebrates or complex invertebrates but rather has its molecular origins at least as far back as the simplest unicellular eukaryotes. The present data, however, demonstrate a limited proteolysis of insulin by fungal carboxyl proteinases from A. sojae and P. coccineus, which are a fungi imperfecti and a wood-deteriorating Basidiomycete, respectively. Based on the present findings, we hypothesize that a degradation of insulin, which structure may be partly dissociated as a monomer with a molecular weight of 6000, is effected in vivo by a nonspecific carboxyl proteinase such as cathepsin D (EC 3.4.23.5). Furthermore, we hypothesize that a degradation of an

'immunologicaUy and biologically active insulin' in unicellular eukaryote [8] is effected by fungal carboxyl proteinases such as A. sojae carboxyl proteinase I and P. coccineus carboxyl proteinase I a•

References I Duckworth, W.C. and Halban, P.A. (1980) Diabetologia 19, 270-271 2 Duckworth, W.C., Heinemann, M. and Kitabchi, A.E. (1972) Proc. Natl. Acad. Sci. USA 69, 3698-3702 3 Duckworth, W.C., Stenz, F.B., Heinemann, M. and Kitabchi, A.E. (1979) Proc. Natl. Acad. Sci. USA 76, 635639 4 Kimura, T., Mayumi, H., Takeuchi, M., Hayashi, K. and Ichishima, E. (1979) Curr. Microbiol. 3, 153-156 5 Reichelt, D., Jacobson, E. and Haschen, R.J. (1974) Biochim. Biophys. Acta 341, 15-26 6 Ichishima, E., Kumagai, H. and Tomoda, K. (1980) Curr. Micrbiol. 3, 333-337 7 Kumagal, H., Matsue, M., Majima, E., Tomoda, K. and Ichishima, E, (1981) Agile. Biol. Chem. Toyko 45, 981-985 8 Roith, D.L., Slailoach, J., Roth, J. and Lesniak, M.A. (1980) Proc. Natl. Acad. Sci. USA 77, 6184-6188 9 Yokoyama, S., Oobayashi, A., Tanabe, O. and Ichishima, E. (1974) Appl. Microbiol. 28, 742-747 10 Tanaka, N., Takeuchi, M. and Ichishima, E. (1977) Biochim. Biophys. Acta 485, 406-416 II Craig, L.C., Konigsberg, W. and King, T.P. (1961) Biochem. Prep. 8, 70-75 12 Frankel-Conrat, H., Harris, J.I. and Levy, A.L. (1955) in Methods in Biochemical Analysis (Glick, D., ed.), Vol. 2, pp. 359-425, Interscience, new York 13 Lowther, A.G. (1951) Nature 167, 767-768 14 Yokoyarna, S., Oobayaslai, A., Tanabe, O. and Ichishima, E. (1975) Biochim. Biophys. Acta 397, 443-448 15 Yokoyama, S., Oobayashi, A., Tanabe, O., Ohata, K., Shibata, Y. and Ichishima, E. (1975) Experimentia 31, 1122-1123 16 Yokoyama, S., Miyabe, T., Oobayashi, A., Tanabe, O. and Ichishima, E. (1981) Agric. Biol. Chem. Tokyo 45, 311-312 17 Li, C.H. (1954) in The Proteins (Neurath, H. and Baily, K., eds.), Vol. 2, pp. 634-650, Academic Press, New York 18 Blundell, T., Dodson, G., Hodgkin, D. and Mercola, D. (1972) in Advances in Protein Chemistry (Anfinsen, C.B., Edsall, J.T. and Richards, F.M., eds.), Vol. 26, pp. 278-402, Academic Press, New York 19 Doty, P., Gellart, M. and Rabinowich, B. (1952) J. Am. Chem. Soc. 74, 2065-2069 20 Frederiq, E. and Neurath, H. (1950) J. Am. Chem. Soc. 72, 2684-269 I 21 Schechter, I. and Berger, A.C. (1967) Biochem. Biophys. Res. Commun. 27, 157-162 22 Ryle, A.P., Leclerc, J. and Falla, F. (1968) Biochem. J. 110,

4p 23 Notkins, A.L. (1979) Sci. Am. 241, 56-67 24 Owerbach, D., Bell, G.I., Rutter, W.J. and Shows, T.B. (1980) Nature 286, 82-84