INACTIVATION OF HUMAN PEPSIN AND GASTRICSIN
Comparative Studies on the Effect of Specific Inactivators on Human Gastricsin and Pepsin* Mike Hunkapiller,t John E. Heinze,f and John N. Mills
ABSTRACT : The
human gastric proteolytic enzymes, gastricsin and pepsin, are known to have different specificities. A comparative study of the effects of several specific inactivators of porcine pepsin has been made to determine differences in the chemical nature of the enzymes' active sites. Inactivators used in this study include NaAc-DL-NleOMe, 2-diazo-4 '-bromoacetophenone (DBA), and 2,4 '-dibromoacetophenone (BBA). Activity of the two human enzymes, measured by hydrolysis of both hemoglobin and synthetic dipeptide substrates, can be completely eliminated by either NnAc-DL-NleOMeor DBA. However, the reactions with gastricsin are slower than those
I?
epsin and gastricsin are proteolytic enzymes known to be present in human gastric juice (Richmond et a/., 1958). Human pepsin has been shown to be similar to porcine pepsin in several physical and chemical properties (Mills and Tang, 1967). By the same criteria, human gastricsin is quite different. Of particular interest is the report that the specificity of gastricsin is similar to, but not identical with, that of pepsin. Both human and porcine pepsin hydrolyze N-Ac-L-Phe-L-I*Tyr, but gastricsin does not (Chiang et a/., 1966). On the other hand, gastricsin hydrolyzes certain peptide bonds involving tyrosine and tryptophan which are not hydrolyzed by human and porcine pepsins (Huang and Tang, 1969a). Nevertheless, an amino acid sequence homology between gastricsin and pepsin strengthens the argument that the active centers may be similar (Huang and Tang, 1969b, 1970). Specificity differences noted above suggest that they are not identical. A study of the effect of several specific inactivators of porcine pepsin on human pepsin and gastricsin might be expected to yield some information about the similarities and differences in the respective active centers and the mechanisms of action of the enzymes. Several specific inactivators of porcine pepsin have been reported. 2,4 '-Dibromoacetophenone (p-bromophenacyl bromide) and related a-halo ketones inactivate porcine pepsin by esterification of the P-carboxyl group of an aspartyl residue on the enzyme (Erlanger et al., 1965, 1966). Several a-diazo ketones also inactivate porcine pepsin, but they react with a different aspartyl residue. Among these compounds are 2-diazo-4 '-bromoacetophenone (Erlanger et a/., 1967); NzAc-
* From the Department of Chemistry, Oklahoma Baptist University, Shawnee, Oklahoma 74801. Received March 30, 1970. This study was supported by National Institute of Arthritis and Metabolic Diseases G r a n t A M 12617. t Present address: Department of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, Calif. $ Present address: Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Ill.
under similar conditions with the two pepsins and show a slightly different pH dependence. BBA, which decreases the hemoglobin-hydrolyzing activity of pepsin by 75-80 %, has no effect under similar conditions on the proteolytic activity of gastricsin. Amino acid analyses of pepsin and gastricsin inactivated by NAc-DL-NleOMe show that complete inactivation is achieved by reaction of one molecule of N,Ac-DLNleOMe per molecule of enzyme. Using results of these inactivation studies, a comparison is made of the active sites of pepsin and gastricsin, and a possible explanation of differences in specificity is given.
DL-NleOMe (Rajagopalan et a[., 1966; Lundblad and Stein, 1969); and NzAcGlyOEt (Kozlov et al., 1967). These inactivators, since they are relatively specific, provide a means for comparative study of their effect on human pepsin and gastricsin. The current study indicated that the diazo ketones, in the presence of Cu(II), inactivate both human pepsin and human gastricsin, but that the halo ketones inactivate only pepsin. Materials Human pepsin and gastricsin were prepared as described previously by Richmond et a/. (1958), rechromatographed on a column of Amberlite CG-50, and further purified by gel filtration on Sephadex G-75 (Mills and Tang, 1967). Porcine pepsin (three-times crystallized) and bovine hemoglobin were obtained from Nutritional Biochemicals Corp. (Cleveland). N-Ac-L-Phe-L-InTyr and N-Cbz-L-Tyr-L-Ala were purchased from Cyclo Chemicals Corp. (Los Angeles). Amberlite CG-50 was obtained from Mallinckrodt (St. Louis). Sephadex G-25 and G-75 were purchased from Pharmacia (Upsala, Sweden). BBAl and BCA were obtained from Matheson, Coleman and Bell (Cincinnati). The diazo compounds were synthesized in our laboratories according to procedures described by Rajagopalan et al. (1966) for N2AcDL-NleOMe, Erlanger et al. (1967) for 2-diazo-4'-bromoacetophenone, and Kozlov et al. (1967) for NzAcGlyOEt. Methods Proteolytic Activity. The procedure of Anson and Mirsky (1932) was used. The hydrolysis of acid-denatured bovine hemoglobin at pH 1.8, 37", was followed by measuring the 280-mp absorbance of trichloroacetic acid soluble hydroly-
* The abbreviations used are these : BBA, 2,4'-dibromoacetophenone; BCA, 2-bromo-4'-chloroacetophenone; DBA, 2-diazo-4'-bromoacetophenone. B I O C H E M I S T R VYO, L . 9,
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3 : Inactivation of gastric proteolytic enzymes by 2-diazo-4‘bromoacetophenone. Inactivation was carried out with 0.015 mM enzyme, 0.04 M sodium acetate buffer (pH 5.6), 25”, 0.50 mM Cu(II), 10% methanol, 0.30 mM DBA. FIGURE
1: Inactivation of gastric proteolytic enzymes by 2,4’dibromoacetophenone. Inactivation was carried out with 0.015 mM enzyme, 0.10 M citric acid-NazHPOr buffer (pH 2.5), 25”, 10% methanol, 0.30 mM BBA.
FIGURE
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Effect of pH on inactivation of gastric proteolytic enzymes by 2,4’-dibromoacetophenone.Conditions were same as those described in Figure l, with 0.05 M HCl-KCl or 0.10 M citric acid buffers: time of inactivation (hr): porcine pepsin (6); human pepsin (18); human gastricsin (18). FIGURE 2:
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sis products in a Zeiss PMQ I1 spectrophotometer. Assays were always run in duplicate, and the average deviation was *3%. Activity toward Synthetic Substrates. Hydrolysis of N-AcL-Phe-L-12Tyr and N-Cbz-L-Tyr-L-Ala was measured by the increase in ninhydrin color after hydrolysis at p H 2.0, 37O, as described previously by Chiang et al. (1966). Assays were always run in triplicate, and the average deviation was i5 %. Znactiuation Studies. The procedures were essentially those used in the previously cited studies with porcine pepsin (Lundblad and Stein, 1969). In a typical inactivation study, 0.50 ml of methanol containing the inactivator was added with shaking to 4.5 ml of enzyme solution in an appropriate buffer (see legends to figures). Unless otherwise noted, final enzyme concentration was 0.050 (0.01 5 mM) and inactivator concentration was 0.30 mM. Cupric acetate was present at 0.50 mM concentration in studies with diazoketones. At selected times, 50-pl aliquots were withdrawn for activity assay. Controls in which 0.50 ml of methanol and/or cupric acetate solution were added to the enzyme solution were always run, and their activity was taken as 100%. At the concentrations used, these reagents alone did not affect enzymic activity of either pepsin or gastricsin. Stoichiometry 0.f Inactivation by Diazoacetyl-DL-norleucine Merliyl Ester. Incorporation of norleucine was measured as described by Rajagopalan et al. (1966). Amino acid analyses were performed according to the method of Spackman el al. (1958) with a Spinco amino acid analyzer, Model 1208. Enzyme samples (2.5 mg) were hydrolyzed by 1.O ml of twiceglass-distilled HCl a t 110 i 2 ” for 24 hr.
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INACTIVATION OF HUMAN PEPSIN AND GASTRICSIN
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Effect of pH on inactivation of gastric proteolytic enzymes by 2-diazo-4'-bromoacetophenone,Conditions were the sameas those described in Figure 3, with 0.04 M sodium acetate buffers; time of inactivation (min): porcine pepsin (2); human pepsin (2); human gastricsin (20).
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FIGURE 5 : Effect of prior mixing of Cu(I1) and diazoacetyl-DLnorleucine methyl ester on rate of inactivation of gastric proteolytic enzymes. Inactivation was carried out with 0.015 mM enzyme, 0.04 M sodium acetate buffer (pH 5.6), 25", 10% methanol, 0.50 KIM Cu(II), 0.30 mM NnAc-DL-NleOMe.For prior mixing reactions, equal volumes of methanolic solutions of 1.00 mM Cu(I1) and 0.60 mM NaAc-DL-NleOMewere mixed for 10 min prior to addition to enzyme.
Results Inacfiwtion b y a-Haloketones. Figure 1 shows the rates of inactivation of human pepsin, human gastricsin, and porcine pepsin by BBA. Although the rate of inactivation of human pepsin is somewhat less than that of porcine pepsin, the maximum per cent of inactivation (75-80%, hemoglobin assay) is the same for both enzymes. In the same experiment, decrease of N-Ac-L-Phe-L-I,Tyr hydrolyzing activity reached a maximum of 60-65 % for pepsins. Figure 2 shows plots of the pH dependence of the rate of inactivation by BBA. The broad maximum between pH 2 and 3 is similar to that obtained by Erlanger et al. (1965) with porcine pepsin. Figures 1 and 2 also show that no inactivation of hemoglobin-hydrolyzing activity of human gastricsin is found even after 48 hr of incubation with a 20-fold molar excess of BBA. The same result is obtained when gastricsin activity is measured by hydrolysis of N-Cbz-L-Tyr-L-Ala.Increasing the BBA concentration to a 200-fold molar excess also has no effect on gastricsin activity. BCA is as potent an inactivator of human pepsin as is BBA. Maximum inactivation at pH 2.5, 25", with a 20-fold molar excess of reagent was 70-75 % (hemoglobin assay). N o inactivation of gastricsin was found. When 0.15 mM human pepsin is treated with a 10% molar excess of BBA at pH 2.5,25", for 48 hr, the extent of inactivation (70-75%, hemoglobin assay) closely approaches that for the reaction with a 20-fold molar excess of reagent. This suggests that the incorporation of 1 mole of thep-bromophenacyl
moiety per mole of pepsin is responsible for the inactivation, but we have not yet confirmed this by bromine analysis. If a solution of human pepsin inactivated by BBA at pH 2.5 is brought to pH 5.3 and a 33-fold molar excess of Cu(I1) and a 20-fold molar excess of N2Ac-m-NleOMe are added, the remaining hemoglobin-hydrolyzing activity (20-25 %) is lost after 15 min with the incorporation of one residue of norleucine per molecule of pepsin. Lundblad and Stein (1969) and Erlanger et al. (1967) observed the same result with porcine pepsin. Inactivation by a-Diaroketones. In the presence of Cu(II), DBA and the inactivators synthesized from dipeptides completely eliminate both the proteolytic (hemoglobin hydrolyzing) activity of human pepsin and gastricsin and their peptidase (pepsin N-Ac-L-Phe-L-I,Tyr hydrolyzing and gastricsin N-Cbz-L-Tyr-L-Ala hydrolyzing) activity. Rates of inactivation of the two pepsins are very similar, while inactivation of gastricsin is always slower. The differencein rate of reaction in DBA is shown in Figure 3. Inactivation of the pepsins is essentially complete after 10 min, while complete inactivation of gastricsin requires 45 min. The pH-rate profile for inactivation by DBA (Figure 4) also shows similarity of the two pepsins and a slightly different pH dependence for gastricsin. All three enzymes show a maximum rate of inactivation at pH 5.3. Of note is the much increased rate of inactivation when methanolic solutions of Cu(I1) and NzAc-m-NleOMe are
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6: Effect of prior mixing of Cu(I1) and diazoacetyl-DLnorleucine methyl ester on pH dependence of inactivation of gastric
FIGURE
proteolytic enzymes, Conditions were the same as those described in Figure 5 , with 0.04 M sodium acetate buffers; time of inactivation (min): human pepsin without prior mixing (15); human pepsin with prior mixing (2); human gastricsin without prior mixing (25); human gastricsin with prior mixing (5).
mixed prior to addition of the enzyme (Figure 5). Lundblad and Stein (1969) suggest that Cu(I1) and N2Ac-DL-NleOMe form a highly reactive intermediate. Our work with human gastricsin and pepsin supports this idea. Prior mixing of Cu(I1) and NzAc-DL-NleOMe(15 min, 25 ") accelerates inactivation by a factor of 8 for pepsin and 4 for gastricsin (based on time of complete inactivation), Mixing of Cu(I1) and enzyme for 15 min prior to addition of N2Ac-DL-NleOMeor mixing of N2AcDL-NleOMe and enzyme prior to addition of Cu(I1) causes no change in the rate of inactivation compared to that obtained when all three reagents are added simultaneously. Figure 6 shows the pH-rate profile for inactivation by NzAc-m-NleOMe. Without prior mixing, the rates of inactivation show a steady increase from pH 4 to 6 (a study of the inactivation by diazoketones at pH above 6 is complicated by denaturation of the enzymes in neutral or alkaline range; however, they were stable under conditions used for inactivation studies reported in this paper). With prior mixing of Cu(1I) and N&-DL-NleOMe, sharp maximum rates of inactivation are observed at pH 5.3, the same optima shown for inactivation by DBA without prior mixing (Figure 4). Lundblad and Stein (1969) suggest that the pH curve obtained without prior mixing is partly an indication of the pH dependence of the formation of the reactive intermediate. Figure 7 shows that Cu(I1) is essential for the inactivation of human gastricsin by N2AcGlyOEt. As the concentration of Cu(I1) is decreased from 3.00 to 0.25 mM (gastricsin concen-
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7: Effect of concentration of Cu(I1)on inactivation of human gastricsin by diazoacetylglycine ethyl ester. Inactivation was carried out with 0.015 mM gastricsin, 0.04 M sodium acetate buffer (pH 5.6), 25', 10% methanol, 0.30 mM N,AcGlyOEt. Molar ratios of Cu(I1) to gastricsin are shown in the figure.
FIGURE
tration maintained at 0.015 mM), the rate of inactivation steadily decreases. In the absence of Cu(II), inactivation by 0.30 mM NzAcGlyOEt amounts to only 2-3% after 30-min incubation. Lundblad and Stein (1969) found similar results with porcine pepsin. Stoichiometry of' Inactiaation by N2Ac-~~-NleOMe. Amino acid analyses of human pepsin and gastricsin inactivated by a 20-fold molar excess of NzAc-DL-NleOMe at pH 5.6 show 1.06 residue of norleucine per gastricsin molecule of 298 amino acid residues (Mills and Tang, 1967) and 1.14 residue of norleucine per pepsin molecule of 337 amino acid residues (Mills and Tang, 1967). Rajagopalan et al. (1966) found 0.94 norleucine residue incorporated per porcine pepsin molecule of 321 amino acid residues. Discussion It appears certain that the nature of the active centers as well as the catalytic mechanisms of gastricsin and pepsin are similar. Both enzymes are inactivated by the same compounds, the a-diazoketones. Cu(I1) is essential for inactivation of both enzymes. Inactivation (100%) is observed when enzymic activity is measured by either hemoglobin assay or synthetic dipeptide substrate assay, Moreover, complete inactivation by N2Ac-~~-NleOM requires e the incorporation of only 1 equiv of norleucine into either enzyme. This indicates that in both cases inactivation is due to a specific reaction at or near the active site of the enzyme. The above observations suggest that the active centers, and possibly the catalytic mechanisms, of
INACTIVATION OF HUMAN PEPSIN AND GASTRICSIN
TABLE I: Summary of Inactivation of Human Pepsin and Gastricsin by Halo
Ketones and Diazo Ketones,
Time of Incubation with Inactivator
Per Cent Inactivation of HemoglobinHydrolyzing Activity Human Pepsin
Porcine Pepsin
8 hr 48 hr
40 76
62 78
0.00
8 hr 48 hr
35 73
59 75
5.6
0.50
5 min 15 min
51 70
95 100
95 100
NzAc-DL-NleOMe
5.6
0.50
5 min 15 min
2 8
37 86
40 88
NzAc-DL-NleOMe (prior mixing)
5.6
0.50
5 min 15 min
54 100
100 100
100 100
NiAcGly OEt
5.6
0.50
5 min 15 min
40 96
100
100
100
100
NzACGl y OEt DBA NAc-DL-NleOMe
5.6
0.00
1 hr
1
2
4
Inactivator
PH
Cu(I1)
BBA
2.5
0.00
BCA
2.5
DBA
human pepsin and gastricsin are similar in nature. This agrees with observations on the similarity in specificity (Huang and Tang, 1969a) and amino acid sequence (Haung and Tang, 1969b, 1970). The difference in rates of inactivation of pepsin and gastricsin by the diazoketones (summarized in Table I) may be due either to steric effects of the active sites of the enzymes or to the reactivity of the respective functional groups. Steric effects may also account for the absence of the inactivation of gastricsin by the a-haloketones. An alternative interpretation would be that gastricsin lacks a group (presumably a carboxyl group) suited chemically and/or sterically for reaction with the haloketones. Erlanger et al. (1966, 1967) showed that inactivation of porcine pepsin by haloketones was due to steric hindrance of substrate binding caused by esterification of the /3-carboxyl group of an aspartyl residue. It is conceivable that the haloketones could react with a carboxyl group on the gastricsin molecule without affecting activity. However, in view of the specificity of reaction (BBA apparently reacts with only one of the acidic residues on either human or porcine pepsin), this seems unlikely. We have not been able to demonstrate any significant difference between the inactivation reactions of human and porcine pepsin by either the diazoketones or the haloketones. In view of this and the physical and chemical similarities of the two enzymes (Mills and Tang, 1967) and the amino acid sequence homology reported by Huang and Tang (1969b, 1970), it seems reasonable to conclude that the active sites of the enzymes from the two species are very similar. Lundblad and Stein (1969) considered kinetic and inactivation studies and implicated several carboxyl groups, each with a different pK, in the catalytic function of porcine pepsin.
Human Gastricsin
They proposed that inactivation by the diazoketones proceeded via a metal-complexed carbene. This positively charged intermediate would be directed to the active site by an ionized carboxyl group with a pK near 4 and would esterify a protonated carboxyl group with a higher pK. If the same reaction mechanism occurs in the inactivation of gastricsin, as is suggested by our results, a partial explanation for the higher pH optimum for proteolysis by gastricsin is possible. The results shown in Figures 4 and 6 would indicate that the inactivatordirecting ionized carboxyl group has a slightly higher pK than the corresponding group in pepsin. If the carboxyl groups are indeed essential for catalysis, their pK's would influence their optimum pH for catalysis. Moreover, the nearness to thc active site of porcine and human pepsin of a reactive carboxyl group whose esterification by haloketones partially blocks enzymic activity indicates that this carboxyl group could easily help or hinder the binding and hydrolysis of substrates. The fact that gastricsin apparently does not have a group susceptible to such esterification suggests that gastricsin should be capable of binding and hydrolyzing a slightly different set of substrates. Again, differences in chemical environment of the active sites can explain the different specificities of human pepsin and gastricsin. Acknowledgments We wish to thank Dr. Jordan Tang for serving as a consultant on this project; Dr. R. G . Canham for several helpful suggestions; the Biology Department of Oklahoma Baptist University for use of their equipment; Drs. Stanford Moore and R. L. Lundblad for helpful advice on the preparation of NzAc-m-NleOMe; and the Neurosciences Section of the
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Oklahoma Medical Research Foundation for use of their amino acid analyzer. Special thanks go to Mrs. Vivian Davis for performing the amino acid analyses. References Anson, M. L., and Mirsky, A. E. (1932), J . Gen. Physioi. 16, 59. Chiang, L., Sanchez-Chiang, L., Wolf, S., and Tang, J. (1966), Proc. Soc. Exp. Biol. Med. 122,700. Erlanger, B. F., Vratsanos, S. M., Wassermann, N., and Cooper, A . G. (1965), J . B i d . Chem. 240, PC3447. Erlanger, B. F., Vratsanos, S. M., Wassermann, N., and Cooper, A. G. (1966), Biochem. Biophys. Res. Commun. 23,243. Erlanger, B. F., Vratsanos, S. M., Wassermann, N., and
e t a/.
Cooper, A. G. (1967), Biochem. Biophys. Res. Commun. 28,203. Huang, W. Y . ,andTang, J. (1969a), J . Biol. Chem. 244,1085. Huang, W. Y . ,andTang, J. (1969b), Fed. Proc. 28,662. Huang, W. Y . ,and Tang, J. (1970), J . Biol. Chem. (in press). Kozlov, L. V., Ginodman, L. M., and Orekhovich, V. N. (1967), Biokhimiya 32, 1011. Lundblad, R. L., and Stein, W. H. (1969), J . Biol. Chern. 244, 154. Mills, J. N., andrang, J. (1967), J. Biol. Chem. 242, 3093. Rajagopalan, T. G., Stein, W. H., and Moore, S. (1966), J . Biol. Chem. 241,4295. Richmond, V., Tang, J., Wolf, S., Trucco, R., and Caputto, R. (1958)) Biochim. Biophys. Acta 29,453. Spackman, D. H., Stein, W. H., and Moore, S. (1958), Anal. Chem. 30, 1190.
Bacterial Bioluminescence. Comparisons of Bioluminescence Emission Spectra, the Fluorescence of Luciferase Reaction Mixtures, and the Fluorescence of Flavin Cations" Michael Eley,t John Lee, J.-M. Lhoste, C. Y . Lee, Milton J. Cormier,$ and P. Hemmerich
ABSTRACT : The fluorescence
emission spectra of flavin cations in several rigid solvents were measured. Solvent polarity conditions were found in which these fluorescence spectra closely match the emission spectra of bacterial bioluminescence for the in aim systems, the in zitro reaction initiated with various re-
I
t has long been known that the requirements for cell-free bacterial bioluminescence are bacterial luciferase (Strehler, 1953)) reduced FMNl and oxygen (Strehler et ai., 1954), and a long-chain fatty aldehyde (Cormier and Strehler, 1953). Studies on pure crystalline luciferase isolated from Phorobacterium Jischeri (Kuwabara et al., 1965; Cormier and Kuwabara, 1.965; Hastings et a/., 1969) have not established ~-
* From
the Department of Biochemistry, University of Georgia, Athens, Georgia 30601 ; New England Institute, Ridgefield, Connecticut 06877; M u i u m National d'Histoire Naturelle, Paris 5 , France; Universitat Konstanz, Konstanz, Germany. Receiced March 23, 1970. This work was supported in part by grants from the U. S. Atomic Energy Commission [AT(40-1)-2741, AT (30-1)3401, and AT(40-1)-3974] and the National Science Foundation (GB-7400x1). M. J. C. is a NIH Career Development Awardee (201
