Isolation and properties of ovine [1-pyroglutamic acid]-β-lipoprotein

124

Biochimica et Biophysica Acta, 451 (1976) 124--132

© Elsevier/North-Holland Biomedical Press

BBA 28083 ISOLATION AND PROPERTIES OF OVINE [I-PYROGLUTAMIC ACID ]-/~-LIPOPROTEIN

DONALD YAMASHIRO and CHOH HAO LI The Hormone Research Laboratory, University o f California, San Francisco, Calif. 94143 (U.S.A.)

(Received May 31st, 1976)

Summary A new form of ovine ~-lipotropin has been isolated by partition chromatography on agarose gel. All the evidence is consistent with the conclusion that its structure is identical to that of ovine ~-lipotropin with the exception that the glutamic acid residue in position 1 was replaced by a pyroglutamic acid residue. Its lipolytic activity in isolated rabbit fat cells was about one-half that of ~-lipotropin.

The lipotropic hormone was first isolated from sheep pituitary glands [1,2] and subsequently from those of bovine [3], porcine [4--6] and human [7] origin. In each case only one form of the hormone has been detected, and complete primary structures for the ovine (Fig. 1 and 8--10) porcine [11--13] and human [14] hormones have been elucidated. During investigations on the partition chromatography of proteins on agarose gel, another form of the ovine hormone was discovered. This paper reports the isolation and properties of ovine [ 1-pyroglutamic acid] -fl-lipotropin. Experimental 2-Butanol azeotrope was prepared by distillation from a mixture of 1435 ml of 2-butanol, 550 ml of water, 15 g of zinc dust, and 10 g of tris (hydroxymethyl)-aminomethane. Thin-layer chromatography was run on silica gel in 1butanol/acetic acid/water, 4 : 1 : 1 (solvent 1), and in 1-butanol/pyridine/acetic acid/water, 30 : 20 : 6 : 24 (solvent 2). Electrophoresis on Whatman 3 MM paper was carried out at 400 V for 4--6 h at pH 6.7 in 7-collidine (8.9 ml)/acetic acid (3,1 ml) diluted to 1 1 with water and at pH 3.7 in pyridine (3.3 ml)/ A b b r e v i a t i o n s : < G l u ~ p y r o g l u t a m y l r e s i d u e . O t h e r a b b r e v i a t i o n s are i n a c c o r d a n c e w i t h t h e r e c o m mendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1971).

125 acetic acid (33.3 ml) diluted to a 1 with water. Total enzyme digestion was performed with a mixture of trypsin (Serva) and chymotrypsin (Worthington) in 0.05 M Tris buffer (pH 8.5, 10 mM Mg 2÷) at 37°C for 24 h; the solution was heated at 100°C for 20 min, then treated with leucine aminopeptidase (Worthington) at 37°C for 24 h. Amino acid analysis was performed by the method of Spackman et al. [15]. Amino end group determination was performed by the dansyl method [16,17]. fl-lipotropin was isolated from fresh sheep pituitary glands by the procedure previously described [8]. Lipolytic activity in rabbit fat cells was measured by procedures detailed elsewhere [18], based on methods of Rodbell [19] and Vaughan [20].

Isolation of < GIu-[J-lipotropin by partition chromatography on agarose gel Agarose gel (Bio-Gel A-5m, 200--400 mesh, Bio-Rad Labs) was used as received. The gel slurry was deaerated at the water pump and packed in a glass column (1.05 cm internal diameter) on a pyrex glass wool support in water to a height of 15--20 cm. The length of the column was limited due to the high resistance to flow offered by agarose gel. The solvent system was prepared by mixing 2-butanol azeotrope (150 ml, deaeraLed), water (97 ml, deaerated), glacial acetic acid (4.3 ml), 10% (w/w) aqueous trichloracetic acid (7.5 ml), and sodium chloride (4.4 g). The column was equilibrated first with the lower phase (65 ml) overnight, then with the upper phase with gradually increasing pressure till the upper phase emerged in the eluate. The hold-up volume (VH) is defined as the volume of aqueous phase displaced from the gel bed by the organic phase. A sample (1--10 mg) of ~-lipotropin was dissolved in 0.5 M acetic acid (50 pl}, diluted with the upper phase of the solvent system (0.25--0.50 ml), and applied to the column. Elution with the upper phase was performed (20-30 cm positive hydrostatic pressure from the reservoir) at flow rates of 1--2 ml per h with collection of 0.40-ml fractions. The chromatogram was delineated by the Folin-Lowry method [21]. For isolation of the proteins (Components A and B, see Fig. 2) in the eluates, appropriate tubes were pooled, mixed with about an equal volume of water, and dialyzed at 4°C first against 0.1 M NH4HCO3 then against water. The dialyzed material was submitted directly to chromatography on a 1.05 X 24 cm column of carboxymethylcellulose (125 ml mixing chamber) by procedures previously described [8]. Thorough dialysis prior to the chromatography was essential, otherwise the proteins eluted with the starting buffer. The agarose gel was discarded after an experiment since its reuse was not thoroughly investigated.

Synthesis of
and H-Glu-Leu-Thr-Gly-Glu-

The solid-phase method [22] was employed by procedures detailed previously [23]. The esterification [24] of Boc-Arg(Tos)-OH with chloromethylated polymer was sluggish, therefore resin (2 g) with a chlorine content of 1.29 mmol/g was used and-the reaction was carried out with 4.75 mmol of the tetramethyl-ammonium salt of Boc-Arg(Tos)-OH in the presence of K I (35 mg) in dimethylformamide (14 ml) at 25°C for 17.5 h and at 55°C for 5.5 h; substitution was 0.24 mmol/g by the Gisin method [25]. Incorporation of the pyro-

126 H-Qlu-Leu-Thr-Gly-Glu-Arg-Leu-Glu-Gln-Ala5

10

Arg-Gly-Pro-Glu-Ala-Gla-Ala-Glu-Ser-Ala15 Z0

A l a - A l a - A r g - Ala- Glu- L e u - Glu- Tyr- G1 y- L e u 25 30

Val- A-la- G l u - A l a - Glu- Ala- Ala- glu- L y s - Lys35 40

A s p - Ser- Gly- P r o - Tyr- Lys- Met- Giu- His - P h e 45

50

Arg-Trp-Gly-Ser-Pro-Pro 55

Lys-Asp-Lys-Arg60

T y r - Gly- Gly- P h e - Met- T h r 65

Se r- Glu- Lys - Ser70

Gln- T h r - P r o - Le u- Val- T hr- L e u- P h e - L y s - A s n75 80

A l a - I1 e - I1 e - L y s - A s n - A l a - H i s - L y s - L y s - G I y - G l n - O H

90

85 Fig. 1.

The amino

acid sequence

of ovine fl-lipotropin.

glutamyl residue in I was accomplished with the pentachlorophenyl ester [26]. After deblocking and cleavage of the peptides by the HF method [27--29], they were purified by gel filtration on Sephadex G-10 in 0.5 M acetic acid and by partition chromatography on Sephadex G-25 in 1-butanol/ethanol/2 M aqueous ammonium acetate (4 : 2 : 5) in which peptide I had Rf 0.17 and peptide II, Rf 0.07. Amino acid analysis (HC1 hydrolysate) of I gave Argo.0 Thr0.9 Glu2.1 Glyl.o Leul.o and II gave Argu0 Thr0.9 Glu2.~ Glyo.9 Leu~.0. Amino acid analysis of a leucine aminopeptidase digest (18 h at 37°C) of II gave Arg~.o Thr0.9 Glu2.~ Glyu0 Leu,.o when digestion was performed at a pepR~ 0 2 8 E

\

04

o

r-.

03

w t..) Z <

0.2

0.1

RF 021

/

VH

< I0

20

30

[

I

40

50

FRACTION

60

70

80

NUMBER

Partition chromatography of ovine ~-lipotropin (4.9 rag) on a 1.05 Fraction volume of 0.4 ml. Detection by the Folin-Lowry method.

Fig. 2.

X 18.7

cm column

o f a g a r o s e gel.

127

®

®

ORIGIN Lys

Glu

0 B A

Position

o oeeI@ @eoo¢o o Ox e 1

2

3

4

©

®00@o 5

6

7

8

9 10

Fig. 3. S c h e m a t i c diagram o f paper electrophoresis o f tryptic digests of C o m p o n e n t s A and B at pH 6.7. D e t e c t i o n : O, ninhydrin; X, Sakaguchi reagent.

tide concentration of 2 mg/ml with a peptide to enzyme weight ratio of 50 : 1. Digestion at a peptide concentration of 0.04 mg/ml and a peptide to enzyme ratio of 20 : 1 gave Thr0.s Glul.0 Gly0.s Leu0.s and no arginine presumably due to resistance of the COOH-terminal half of II, especially Glu-Arg, to hydrolysis at low peptide concentration. Peptide I is unaffected by leucine aminopeptidase as indicated b y paper electrophoresis at pH 6.7. Treatment of I with trypsin and chymotrypsin followed by leucine aminopeptidase gave Argl.0Thr0.9Glul.~Glyl.0. Paper electrophoresis of I and II at pH 6.7 gave Rf 0.24 for both relative to glutamic acid and at pH 3.7 gave Rf 0.15 and Rf 0.34 for I and II, respectively, relative to lysine. Thin-layer chromatography of I in solvent 1 and solvent 2 (chlorine-tolidine reagent for detection) gave Rf 0.12 and R~ 0.35, respectively, while II gave Rf 0.04 and Rf 0.19, respectively (ninhydrin and chlorine-tolidine reagents for detection).

Isolation and identification o f peptide A C o m p o n e n t A (0.83 mg) was treated with trypsin (17 pg) in 0.1 M Tris buffer (43 pl) at 37 ° for 19 h. Paper electrophoresis at pH 6.7 and detection of peptides with limited ninhydrin reagent (0.01% in ethanol) gave the pattern in Fig. 3. The area of the paper corresponding to position 3 had previously been shown to contain a Sakaguchi-positive peptide corresponding in behaviour to I. The peptide was extracted with 2.0 ml of 0.1 M NH4OH. Thin-layer chromatography in solvent 1 gave Rf 0.12 and in solvent 2, Rf 0.35, identical in behavior to I. Paper electrophoresis at pH 3.7 gave Rf 0.11 relative to lysine, identical to I in behavior. Amino acid analysis (HC1 hydrolysate) gave Arg0.9Thr0.sGlu~. 3Glyl.0Leul.0. The peptide was unaffected b y leucine aminopeptidase as was the case with I. End group determination by the dansyl method showed no glutamic acid or glutamine, b u t after treatment with 1 M NaOH for 71 h glutamic acid was detected. Isolation and identification o f peptide B Component B was processed in the same m~anner as Component A to give peptide B which travels in electrophoresis at pH 6.7 at the same rate as II. Thinlayer chromatography in solvent 1 gave Rf 0.03 and in solvent 2, Rf 0.19, corresponding to the behavior of II. Electrophoresis at pH 3.7 gave Rf 0.34 relative

128 to lysine, identical to the behavior of II. Amino acid analysis (HC1 hydrolysate) gave Argl. 2Thr0.9Glu2.3Gly 1.0Leu 1.1. Analysis of a leucine aminopeptidase digest (performed at peptide concentration of 0.04 mg/ml under the same conditions as for II) gave Thr0.gGlul.0Gly0.sLeul.o and no arginine, corresponding to the behavior of II. There was insufficient material to perform the digestion under the more concentrated condition. Results and Discussion The partition chromatography of polypeptides up to the size of adrenocorticotropic hormone can be achieved on Sephadex G-25 [30] and Sephadex G-50 [31]. In order to extend the m e t h o d to larger molecules, a more permeable solid support is desirable. Attempts to utilize Sephadex G-100 and polyacrylamide gel have not been entirely satisfactory. The use of agarose gel for partition chromatography in Albertsson solvent systems [32] has been reported [33], but under the conditions of operation described in this work these highly viscous systems did not give useful flow rates. However, the agarose gels were found to possess favorable mechanical and physical properties when solvent systems well-known in classical multiple extraction methods were employed. The choice of solvent system was in part guided by the recommendations of Dixon [34] as applied in earlier work on Sephadex G-25 [35]. Specifically, an Rf of 0.3 was sought in accordance with the equation: 1 Rf = 1

+ (Vs/VH)

"

(l/K)

Where Vs is the volume of stationary phase, VH is the volume of the mobile phase, K is the distribution constant, and R~ is defined as VH/VE, VE being the elution volume of the peak. The ratio V s / V H is approximately 1.75 for Sephadex G-25 as well as for Sephadex G-50. For 6% agarose gel this ratio is about 2.5 in the solvent system employed in this work. Partition chromatography of ovine fl-lipotropin in a solvent system composed of 2-butanol, acetic acid, trichloroacetic acid, and water gave a single peak with Rf 0.31 with rather severe "tailing" effects. The inclusion of sodium chloride in the solvent system completely altered the behavior of fi-lipotropin as shown in Fig. 2. To demonstrate that the separation was not artifactual, C o m p o n e n t A and C o m p o n e n t B were isolated and separately rechromatographed. Each c o m p o n e n t behaved as it did in the original separation. The weight ratio of A to B was 2 : 3. This partition system on agarose gel was readily reproducible. The resolution of the hormone into two components obviously led to the question of the identity of fi-lipotropin. Both had identical ultraviolet spectra. Each gave a single band on disc gel electrophoresis with similar mobilities. The amino acid compositions of acid and enzyme hydrolysates (Table I) were close enough to each other that they could n o t be distinguished one from another. The electrophoretic patterns at pH 6.7 of e h y m o t r y p t i e digests of the two were identical. The electrophoretie patterns of tryptic digests (Fig. 3) finally revealed a difference between the two (position 3). C o m p o n e n t B generated a ninhydrin-positive spot in position 3 where Component A gave no correspond-

129 TABLE I AMINO ACID ANALYSES OF OVINE ~-LIPOTROPIN COMPONENTS Amino Acid

Calculated c

Trp Lys His Arg Asp Asn Thr Ser Gin Glu Pro Gly Ala Val Met Ile Leu Tyr Phe

1 10 2 5 2 2 4 5 4 12 5 8 13 2 2 2 6 3 3 a b c d

Acid hydrolysate a

Enzyme digest b

A

B

A

10.1 2.1 4.9 4.1

10.1 2.1 5.1 4.0

1.0 9.4 1.9 4.4 2.9

1.2 9.0 1.7 4.3 2.8

3.9 4.5

4.5

14.5

14.2

16.1 5.2 8.1 12.6 1.9 1.9 1.3 a 6.1 3.1 3.2

12.7 4.6 6.7 13.2 3.3 2.0 2.3 6.1 3.2 3.2

12.8 4.5 7.2 13.3 3.0 1.9 2.2 6.6 3.2 3.2

16.4 5.1 7.6 12.8 2.0 1.9 1.3 d 6.2 3.0 3.1

,o}

B

H y d r o l y s i s in c o n s t a n t b o i l i n g HC1 at l l O ° C f o r 24 h. Digestion with trypsin and c h y m o t r y p s i n followed by leucine aminopeptidase. See Fig. 1. T h e l o w v a l u e s c a n be a c c o u n t e d f o r b y t h e p r e s e n c e o f a n Ile-Ile s e q u e n c e , well k n o w n to be r e s i s t a n t to acid h y d r o l y s i s .

ing spot. The former spot gave a positive response to Sakaguchi's reagent, and in the parallel position for Component A a spot also appeared. The peptide in the spot generated by Component B (referred to as peptide B) was isolated, and amino acid analysis of an acid hydrolysate proved it to correspond to the NH2-terminal hexapeptide portion of fi-lipotropin. Similarly, the corresponding peptide (referred to as peptide A) generated by Component A was analyzed, and it too had the same composition. It appeared evident at this point that Component B was fi-lipotropin. It was then postulated that Component A had the same structure with the exception that the NH2-terminal position was occupied by a pyroglutamyl residue. Indeed, Component A was shown to have no end group at all by the dansyl method, whereas Component B had glutamic acid as indicated by a combination of the dansyl method and amino acid analysis of a leucine aminopeptidase digest. Furthermore, under conditions known to open the pyrrolidone ring [36], peptide A gave a NH2-terminal glutamic acid residue. For additional confirmation of the identities of peptides A and B, authentic samples of
130

A

t, B

f 0f

|

C

Fis. 4. P e p t i d e m a p s o f t r y p t i c d i g e s t s ( d e t e c t i o n b y n i n h y d r i n w i t h s p o t s l i g h t l y c i r c l e d w i t h p e n c i l ) : A, C o m p o n e n t A, t i p o f a r r o w i n d i c a t e s p o s i t i o n o f p e p t i d e A ( n i n h y d r i n n e g a t i v e ; S a k a g u c h i p o s i t i v e ) ; B, C o m p o n e n t B, a r r o w p o i n t s a t p o s i t i o n o f p e p t i d e B ( n i n h y d r i n a n d S a k a g u c h i p o s i t i v e ) ; C, M i x t u r e o f d i g e s t s o f t h e t w o c o m p o n e n t s : tip o f t o p a r r o w i n d i c a t e s P o s i t i o n o f p e p t i d e A a n d b o t t o m a r r o w p o i n t s a t p o s i t i o n o f p e p t i d e B. D e v e l o p m e n t of m a p s b y u p p e r p h a s e o f 1 - b u t a n o l ] a c e t i c a c i d / w a t e r (4 : 1 : 5) ( u p w a r d ) f o l l o w e d by" e l e c t r o p h o r e s i s a t p H 6 , 7 , 4 0 0 V, 6 h ( a n o d i c d i r e c t i o n t o right).

a total e n z y m e hydr ol ys at e was so close to that of C o m p o n e n t B that t h e y could n o t be distinguished by this criterion. Yet, it would appear t hat if peptide A f r o m C o m p o n e n t A could n o t be h y d r o l y z e d furt her enzymatically, a difference would have been detected. This discrepancy was settled by treatm e n t o f peptide I under the same conditions of e n z y m e digestion. The amino acids threonine, glycine, glutamic acid and arginine were released, indicating that a c h y m o t r y p t i c break had occurred at the Leu-Thr bond and t hat the resulting tetr apept i de was then h y d r o l y z e d by Ieucine aminopeptidase. Assuming th at the structures of C o m p o n e n t s A and B are identical except for the

131 T A B L E II L I P O L Y T I C A C T I V I T Y OF OVINE fl-LIPOTROPIN AND I-~GIu-fl-LIPOTROPIN

Preparation

Dose (pg/ml)

Glycerol production * (~mol/g of ceHs p e r h)

fl-l~otropin

1.1 3.3

2.58±0.12 3.38±0.12

1-~Glu~-Upotropin**

1.1 3.3

1.40±0.26 3.08±0.09

* Mean + S.E.; d e t e r m i n a t i o n s in triplicate. ** Relative p o t e n c y to fl-lipotropin, 5 1 . 2 % w i t h 95% c o n f i d e n c e limit of 3 4 . 6 - - 6 8 . 8 and k = 0.1

NH2-terminal position, the only differences in the enzyme hydrolysates would then be expected to be single residues in the glutamic acid and leucine contents. It is well known that such differences may not be detectable for amino acids occurring frequently in a protein. Since C o m p o n e n t A had no amino terminus and peptide A behaved like peptide I, peptide A presumably is released by tryptic cleavage from the NH2terminal portion of Component A. Whether or not the remainder of the structure of Component A parallels that of Component B was tested by comparing peptide maps of tryptic digests of the two (Fig. 4). Both exhibited the same patterns with the exception that B generated a ninhydrin positive spot n o t present in A. Furthermore, a peptide map obtained on a mixture of tryptic digests of A and B gave the same ninhydrin pattern as for B alone. The evidence is consistent with the conclusion that Component B is ovine fl-lipotropin and C o m p o n e n t A is
132

Acknowledgments We are indebted to H.L. Aanning and Richard L. Noble for exploratory work on the partition chromatography of proteins. We thank Kenway Hoey, W.F. Hain and Jean Knorr for their skilled technical assistance and Dr. A.J. Rao for the bioassay data. This work was supported in part by the National Institutes of Health GM-2907. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

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