ARCHIVES
OF
BIOCHEMISTRY
Reversible
AND
BIOPHYSICS
Dissociation
of Enzymes Inhibition
PETER
31-38
111,
BERNFELD, Bio-Research
(1965)
at High
and
Their
by Polyanions’
B. J. BERKELEY, Institute, Received
Dilutions
Cambridge, Decmeber
AND
R. E. BIEBER
Massachusetts
15, 1964
Crystalline preparations of aldolase and lactic dehydrogenase from rabbit muscle, of porcine pancreatic or-amylase, and of sweet potato &amylase exhibit a decrease of their specific activities upon dilution. This phenomenon can best be explained by a dissociation of the enzymes into enzymically inactive products. The enzyme concentrations at which specific activities drop to one half of their maximum values are 2, 1.5, 0.005, and 1 pg per milliliter of digest, respectively. Various macromolecular polycations and diaminodecane, as well as proteins acting in this capacity, reverse the dissociation and, hence, are activators of the diluted enzymes. They have no effect on the activity of concentrated enzyme solutions. Certain polycations which thus activate one enzyme do not necessarily have the same effect on another enzyme. Many macromolecular polyanions, notably polysaccharide sulfate esters, polyvinyl sulfate, and polystyrene sulfonate, inhibit the enzymes. This effect depends not only on the presence of electronegative groups in these substances but also on the chemical nature of their negative groups, as well as on the chemical nature and, to a much lesser degree, on the molecular weight of the polyanions. Different enzymes are inhibited to widely varying extents by polyanions of a different nature. Polyanionic enzyme inhibition may be due either to a promoting effect of polyanions on the dissociation of enzymes or to some other mechanism. Polycations, including many proteins, counteract the enzyme inhibition by polyanions.
The dissociation of proteins into subunits and the subsequent reconstitution of the resulting fragments has become a well-established fact during recent years, and an excellent review of this subject has been published by Reithel (1). Many chemical substances have been described to serve as causative agents of the dissociation of proteins in general and of enzymes in particular. A few years before this concept became generally known, it was shown that high dilution could bring about the reversible dissociation of two enzymes into inactive products (Z-5). The enzymes serving in
those studies were highly purified bovine liver and spleen p-glucuronidases and crude bovine testicular hyaluronidase. It was found that the dissociation of these enzymes into enzymically inactive products was associated with at least three other phenomena, namely the reversal of their dissociation by various polycations including proteins which may act in this capacity, and hence the activation of the diluted enzymes of polycations and proteins, further the inhibition of enzyme activity by polyanions and, finally, the irreversible loss of enzyme activity at high dilutions upon contact with solid phases exhibiting rough surfaces. The purpose of the present work was to investigate analogous phenomena in other enzymes. Their selection was governed by the availability of crystalline enzyme preparations and by the existence of assay meth-
1 This investigation was supported in part by the National Science Foundation Research Grant No. G 23772 and by General Research Support Grants Sol-FR-05525 and Sol-FR-05218 from the National Institutes of Health, U. S. Public Health Service. 31
32
BERNFELD,
BERKELEY,
ods suitable at high enzyme dilutions (below 1 pg enzyme per milliliter of digest). This study was extended t’o enzymes of various origins and different modes of action. Thus, rabbit muscle lactic dehydrogenase, rabbit muscle aldolase, porcine pancreatic Lu-amylase, and sweet potato ,&amylase were selected, representing oxido-reductive, desmolytic, and hydrolytic enzymes, as well as enzymes from the animal and vegetable kingdoms. MATERIALS
AND
METHODS
Enzymes. All enzymes were purchased as crystalline products from either the Worthington Biochemical Corporation, Freehold, New Jersey, or the Mann Research Laborat.ories, Inc., New York, New York. Polycations. The source of these substances or their preparation has been previously reported (3, 5). Polyanions. The preparation of polymeric sulfate esters, in particular sulfated polysaccharides, has been described earlier (6-8). Unless stated otherwise, all polyanions used in the present work were identical with, or similar to, those employed previously: Enzyme assays. Lactic dehydrogenase activity was determined according to the method of Kubowitz and Ott (9), as described by Kornberg (lo), by measuring the decrease in optical density at 340 rnp which accompanies the oxidation of DPNH to DPN+ during the simultaneous reduction of pyruvate to lactate. Aldolase activity was measured by the procedure of Taylor et al. (11, 12), and the alkalilabile phosphate, generated during incubation of fructose-l, 6.diphosphate with enzyme in the presence of KCN, was determined according to the method of Fiske and SubbaRow (13). A slight modification of the procedure was made to facilitate the assay at very low enzyme concentrations by changing the amounts and concentrations used as follows: 1.2 ml of 0.01 M substrate solution in glycine buffer, pH 9.0; 1.2 ml enzyme solution appropriately diluted in 0.1 M KCN-glycine buffer, pH 9.0; alkalinization with 0.3 ml of 7.5 N NaOH, after incubation; neutralization with 0.3 ml of 7.5 N HCl, 15 minutes later; deproteinization with 3 ml of 10% trichloroacetic acid; determination of inorganic phosphorus on the maximum possible sample size of 4.6 ml, after filtration. In some experiments, the KCN-glycine buffer was replaced by sodium sulfite solution in glycine buffer, adjusted to pH 9.0. This made it possible to compare the effect of polyanionic inhibitors with that of silver ions.
ilND
BIEBER
The act,ivities of both OL- and p-amylases were measured by determining t,he increase in reducing power of potat,o amylopectin solutions (Ramalin, provided by Stein, Hall & Co., Inc., New York, New York) with the use of the dinitrosalicylic acid reagent at pH 6.9 and 4.8, respectively (14, 15). In the case of @-amylase the specific enzyme activity declined rapidly with time, even during the initial stages of the reaction. The substrate concentration in the digest was increased, therefore, from 0.5 to 2.5%, and act,ivity was determined at levels of substrate degradation ranging from 1 to 10%. Activities were measured for all enzymes after appropriate periods of incubation and on suitable aliquots, depending on the enzyme dilution. All dat,a are expressed herein as specific enzyme activities, i.e., as the changes observed (milligrams maltose liberat,ed for the amylases, milligrams alkali-labile phosphorus produced for aldolase, and decrease in optical density for lactic dehydrogenase) per milligram of enzyme per minute. Contact of dilut,ed enzyme solutions with glass or other materials having rough surfaces was prevented. All enzyme assays and all enzyme dilutions were carried out in Nalgene or polyethylene tubes which were frequent.ly soft.ened by treatment with boiling water to maintain scratch-free surfaces (3). Only silicone-coated pipettes were used to transfer enzyme solutions. All enzymes were kept in concent.rated solutions (l-10 mg per milliliter) in a household refrigerator. Aliquots of these stock solutions were diluted each day immediately before the experiment. Diluted enzyme solutions were not kept more than a few hours. RESULTS
All four enzymes tested exhibited considerable losses of specific activity upon dilution (Fig. 1). The enzyme concentration at which this phenomenon became effectual was characteristic for each enzyme and varied considerably from one enzyme to another. Porcine pancreatic amylase was far more resistant against dilution than the other three enzymes tested, Table I shows the concentrations at which the specific enzyme activities dropped to one half of their full values. Cysteine had no effect on the loss of specific activit’y of /3-amylase. A number of substances with the common property of being polycations, mostly but not exclusively of macromolecular nature, prevented the loss of specific activity upon dilution of ,&amylase (Table II) ; bovine serum albumin or ovalbumin had the same
REVERSIBLE
DISSOCIATION
a-AhWLASE 9 with OVA q
without
”
OF
ENZYMES
AT
HIGH
33
DILUTIONS
&AMYLASE o with CYST o withwt ”
400
ENZYME
CONCENTRATION
( pq /ml
DIGEST)
FIG. 1. Specific activities (ordinate) of four enzymes, as a function of enzyme concentration (abscissa). Specific activities are expressed as follows: 01- and @-amylases, in mg of maltose/mg of enzyme/minute; aldolase, in mg of alkali-labile phosphate/mg of enzyme/minute times lo3 (in the presence of KCN); lactic dehydrogenase (LDH) in decrease of optical density at 340 mp/minute/mg of enzyme. Porcine pancreatic a-amylase, in the absence or presence of crystalline ovalbumin (OVA), 5 mg/ml; sweet potato @-amylase, in the absence or presence of cysteine (CYST) 20 rg/ml; rabbit muscle aldolase, in the absence or presence of crystalline bovine serum albumin (BSA), 5 mg/ml; rabbit muscle lactic dehydrogenase (LDH), in the absence or presence of crystalline bovine serum albumin (BSA), 5 mg/ml.
TABLE
I
CONCENTRATIONS OF ENZYMES AT WHICH DISSOCIATION TAKES PLACE state of purity of enzyme used
Enzyme
Porcine pancreatic a-amvlase Sweet potato P-amylase Rabbit muscle lactic dehydrogenase Rabbit muscle aldolase Bovine liver or spleen j%glucuronidasea Bovine testicular hyaluronidase* (1 Bernfeld a Bernfeld c Depending
et al. (2). et al. (5). on method
Cont. at which sp. act. has dropped to one half of max. value (J&g/ml)
Cryst.
0.005
Cryst
.
1
Cryst
.
1.5
Cryst.
2
Highly purified
l-2
Crude
of assay
10-100”
used.
effect on the activities of a-amylase, lactic dehydrogenase, and aldolase (Fig. 1). The activation of /3-amylase by protamine sulfate depended chiefly on the enzyme concentra-
tion; activation was most marked at low fi-amylase concentrations and virtually nonexistent at high concentrations (Table III). That the increase of specific activity of /3-amylase by protamine sulfate was not due TABLE INFLUENCE OF POLYCATIONS OF HIGHLY DILUTED
II ON THE ACTIVITY fl-AMYLASE Specific enzyme activityb
Protamine sulfate Poly-r-lysinec Chitosand Polyvinylamine c 1, IO-Diaminodecane None
178 138 145 158 146 34
a At concentrations of 150 pg of activator/ml of digest. * In mg of maltose/mg of enzyme/minute, at 25” in acetate buffer of pH 4.8 and at an enzyme concentration of 0.05 pg/ml of digest. c Hydrobromide, molecular weight 175,000; from Mann Research Laboratories. d Alkali-deacetylated and partially depoly-
merized chi+,in (5, 8).
e By courtesy Kodak Company,
of D. D. Reynolds, Eastman batch J 477-81B, hydrochloride.
BERNFELD,
BERKELEY,
to a stabilization of the enzyme by this polyanion but was a true activation was demonstrated in an experiment in which the activator was added before as well as after incubating the diluted enzyme without substrate (Table IV) ; increase in activity was noted not only when protamine sulfate was added to the enzyme immediately prior to the contact with the substrate (lines A and B) but also when diluted enzyme was first incubated without substrate and then TABLE
III
INFLUENCE OF ENZYME CONCENTRaTION ON ACTIVATION OF p-AMYLUE BY PROT~MINE SULFATE Specifnc
enzyme
activity” In presence of protmine sulfate (150 #g/ml digest)
Without activator
4 2 1 0.5 0.25 0.125 0.0625
THE
235 153 124 102 94 69 49
239 243 234 222 228 234 242
5 In mg of maltose/mg of enzyme/minute, 25” C in acetate buffer of pH 4.8. TABLE INFLUENCE
OF ADDING
LUTED @-AMYLASE, TION, ON THE
Di:h&ing
$tiif
enzymeb
,“~;gc
1 1 1 1
IV THE
POLYANION
TO
DI-
BEFORE OR AFTER INCUBASPECIFIC ENZYME ACTIVITY
Sequence
A B c D
at
2 3
of operations’
Adding substrate
2 3 4 3
Incubating for 1 hour at 2SQC
3 4 2, 5 2, 4
Specific ~IlZyIll~ activityd
113 178 155 98
a The figures indicate in which sequence the operations were performed. b Diluting to a concentration of 0.5 pg/ml. c 150 pg/ml of digest after addition of substrate, or 273 fig/ml of mixture before addition of substrate. d In mg of maltose/mg of enzyme/minute (incubation time after addition of substrate), at 250 C in acetate buffer of pH 4.8 and at an enzyme concentration of 0.25 .ug/ml of digest.
AND
BIEBE
R
brought in contact with protamine sulfate and substrate (lines C and D). Protamine sulfate, poly-n-lysine or 1, lodiaminodecane, at concentrations of 150 Mg per milliliter of digest, could not replace bovine serum albumin as activators of aldolase at the concentration of 3.8 pg enzyme per milliliter of digest. All four enzymes were inhibited at various degrees by polystyrene sulfonate and, to some extent, by other polyanions, such as sulfated polysaccharides. The relationship between enzyme concentration, inhibitor concentration, and specific enzyme activity is exemplified by the inhibition of aldolase by polystyrene sulfonate (Fig. 2) ; for the purpose of comparison, data of the inhibition of this enzyme by silver ions are also given. The inhibition of lactic dehydrogenase and fl-amylase by polystyrene sulfonate, by poly-
‘I
ENZYME CONCENTRATIONS
1 ( pg/ml
INHIBITOR DIGEST 1
FIG. 2. Inhibition of rabbit muscle aldolase by polystyrene sulfonate (Lustrex X770, Monsanto Chemical Company). Left, specific enzyme activity as a function of the enzyme concentration at seven different inhibitor concentrations: curve A 3.3 pg inhibitor/ml; B, 6.6 pg; C, 13.3 pg; D, 26.6 pg; E, 53.5 pg; F, 107 Gg; and G, 213 rg inhibitor/ml of digest; also included are data in the absence of inhibitor (curve 0) and in the presence of 0.33 X 10e3 M silver nitrate (curve Ag). Right, specific enzyme activity as a function of the inhibitor (polystyrene sulfonate) concentration at nine different enzyme concentrations: curve a, 1.2 pg enzyme/ml; b, 2.4 pg; c, 4.8 rg; d, 9.6 rg; e, 19.2 pg; f, 38.4 rg; g, 77 rg; h, 154 rg; and i, 308 rg of enzyme/ml of digest. All enzyme activities were measured in a sodium sulfite-glycine buffer. Both sides of the figure are based on the same set of data which are presented in two different ways. Points of experimental observations are omitted for increased clarity.
REVERSIBLE
DISSOCIATION
OF
vinyl sulfate, and by some sulfated polysaccharides has been found to follow similar patterns. Inhibition of enzymic activity depended significantly on the chemical nature of the
ENZYMES
HIGH
BY POLYANIONS
-
Polystyrene sulfonated Polystyrene sulfonate Polystyrene sulfonate Polystyrene sulfonate Polystyrene sulfonate Polystyrene sulfonatef Polystyrene sulfonate Polystyrene sulfonate Polyvinyl sulfateg Polyvinyl sulfate Polyvinyl sulfate Polyvinyl sulfate Polyvinyl sulfate Sodium dodecylsulfate Sodium dodecylsulfate Sodium dodecylsulfate Dextran sulfate* Dextran sulfate Dextran sulfate Amylopectin sulfate Amylopectin sulfate Amylopect,in sulfate” Amylopect.in sulfate” Amylose sulfate Amylose sulfate” Amylose sulfatei Poly-r-glutamic acidi Polymethacrylic acidk
Degree polymerization
.6708 670 670 670 670
0.64 0.64 0.64 0.64 0.64
1.59 1.59 1.09 1.41 2.13 1.72 1.66 2.11 1.44 1.64 -
Cont. in digest Wml) 500 330 150 50 5 500 330 50 500 330 150 50 5 500 330 50 330 150 150 330 150 330 150 150 150 150 330 330
0.64 0.64 0.64 0.64 0.64
-
-
I-
No. sulfate groups/repeating unit
35
V
INHIBITION
Inhibitor Chemical nature
DILUTIONS
polyanion but not on its degree of polymerization (Table V). Polyanions of low molecular weight, however, such as 4, S-dihydroxy2,7-naphthalene disulfonic acid, had no inhibitory effect . No polyanions containing
TABLE ENZYME
AT
> 6000 > 6000 540 > 2000 > 2000 405 105 -800 160 80 388
-
Per cent inhibition Aldolast?
.-
of enzyme
Lactic dehydrogenaseb 92
88 64 90 60 28 34 0 100 o-7 49 loo 16 51 92 6.4 24 67 46 66 47 53 51 51
-
a 9 pg enzyme/ml of digest, in sulfite-glycine buffer. b 1.4 pg enzyme/ml of digest. c 12.8 pg enzyme/ml of digest, in the presence of cysteine (20 fig/ml). d “Lustrex X770” from the Monsanto Chemical Company, Plastics Division (no longer commercially available); Lustrex X710 with a degree of polymerization of 95” behaved in a similar fashion. e According to New Product Information Bulletin No. D5-lOA, Monsanto Chemical Company. f “Polytak” from the Hagan Chemicals & Controls, Inc .; degree of polymerization unknown. 0 Prepared from commercial polyvinyl alcohol, Elvanol, grade 71/24, DuPont de Nemours & Co. h Prepared from dextran samples provided by Dr. Allene Jeanes; all data on molecular weights and nature or degree of branching of dextran by personal communication of Dr. Jeanes; further details have been published (7, 8). i Prepared by sulfation of the partially hydrolyzed polysaccharide. j Mann Research Laboratories, Inc., molecular weight 50,000. k Sample M-2, obtained from Dr. Herbert Morawetz in 1952.
36
BERNFELD,
BERKELEY,
only carboxyl groups (polyglutamic acid, polymethacrylic acid, Table V) were found to inhibit any of the enzymes under investigaGon. The three enzymes tested behaved in entirely different ways to various polyanions; whereas polystyrene sulfonate inhibited all three enzymes, polyvinyl sulfate affected only t,wo of them to a noticeable degree, i.e., lactic dehydrogenase and @-amylase; the polysaccharide sulfat,es inhibited P-amylase markedly, but the activity of aldolase was hardly affected by them; sodium dodecyl sulfate was a stronger inhibitor of aldolase than of lactic dehydrogenase. These tests were performed at enzyme concentrations sufficiently high to attain near maximum specific enzyme activities (Fig. 1). At lower enzyme concentrations less polyanion was necessary to cause inhibition, whereas much larger amounts of polyanion were required to produce the same effect at higher enzyme concentrations (Fig. 2). In many instances was the inhibition of enzyme activity by polyanions compensated entirely or in part by the addition of polyanions such as protamine sulfate, poly-Llysine, chitosan, polyvinylamine, or 1, lodiaminodecane. Even apparently neutral proteins like bovine serum albumin are known to behave as polycations in this respect and were found to counteract the inhibition of aldolase and lactic dehydrogenase by sodium dodecyl sulfate and other polyanions (Table VI). TABLE VI ACTIVITY OF ALDOLASE IN THE PRESENCE OF SODIUM DODECYL SULFATE AND BOVINE SERUM ALBUMIN Bovine serum albumin (pglml digest)
Sodium dodecyl sulfate (pg/mi digest)
63 125 250 500 500
100 200 400 200 200 200 200 -
110 49.5 38.5 6 44 88 93.5 110 126
a In pg P/pg enzyme/minute; in KCN buffer at an enzyme level of 10.0 pg/ml of digest.
AND
BIEBER DISCUSSION
Evidence for the reversible dissociation of an enzyme into enzymically inactive products under the influence of chemical reagents, such as urea, hydrochloric acid, acetic acid, or sodium dodecyl sulfate, was brought forward by Stellwagen and Schachman (16, 17) and by Deal and Van Holde (18) in the case of rabbit muscle aldolase. These authors were also able to reunite the subunits to the biologically active enzyme. Reversible dissociation under the influence of various chemical reagents has been found for many other enzymes, including alkaline phosphatase (19), glutamic acid dehydrogenase (2023), fumarase (24), lysozyme, Bucillus subtilis cr-amylase (25), bovine pancreatic ribonuclease (26-30), and others (1). The present data clearly demonstrate that high dilution is another factor which may cause the reversible transformation into enzymically inactive products of several crystalline enzymes, namely aldolase and lactic dehydrogenase of rabbit muscle, porcine pancreatic ar-amylaseand sweet potato @-amylase. The reversible loss of specific enzyme activity upon dilution can best be explained by assuming a dissociation of the enzymes into enzymically inactive subunits. Similar findings on the dissociation upon dilution of bovine testicular hyaluronidase (4, 5) and of bovine liver and spleen p-glucuronidases (2, 3) have been reported earlier. A decrease of the molecular weight, as measured by light scattering, of bovine hepatic glutamic acid dehydrogenase, upon the increaseof the dilution, without concomitant loss of enzyme activity was observed by Fisher et al. (23). The present data show that the enzyme concentration, at which the specific activity is reduced and hence dissociat#ion occurs, depends on the nature of the enzyme, as well as, to a large extent, on the state of purit’y of the enzyme (see Table I). Whether dissociation into enzymically active subunits might have occurred at enzyme concentrations higher than those stated in Table I, but might have escapedour attention because only enzyme activities and no molecular weights were measured, cannot be decided from our data. Determinations of rnost physical properties of proteins, leading
REVERSIBLE
DISSOCIATION
OF
to the estimation of their molecular weight, are usually performed at concentrations well above those indicated for the pure enzymes in Table I. Yielding and Tomkins (22) found that n-leucine prevents the dissociation of glutamic acid dehydrogenase into smaller fragments. A somewhat similar phenomenon, described here, is the action of various polycationic substances on the activity of diluted @-amylase, or that of apparently neutral proteins on the activities of diluted a-amylase, lactic dehydrogenase, and aldolase. Likewise, hyaluronidase and @glucuronidase have been reported not to undergo dissociation in the presence of polycations or certain proteins (2, 3, 5). It appears probable that bovine serum albumin, ovalbumin, and possibly also many other proteins with isoelectric points above four behave as polycations in this respect. Since enzymes in biological materials are almost always accompanied by an excess of other proteins, dissociation is unlikely to occur therein, even though the concentration of an individual enzyme might be low enough to cause its dissociation. No explanation can be given at this t’ime for the findings that p-amylase activity at high dilution is preserved by protamine sulfate, polylysine, and several other polycations, as are the activities of hyaluronidase (5) and p-glucuronidase (a), whereas that of aldolase is not affected by these subst’ances but is protected by bovine serum albumin. There is sufficient evidence that the effect of polycations on aldolase is not that of a mere stabilizat’ion of the enzyme against spontaneous loss of activity since the addition of protamine sulfate to the diluted enzyme after incubation without substrate (line C, Table IV) produced a substantial increase of the specific enzyme activity beyond that of the enzyme in the absence of polyanion (line A). The same data also prove that the polycation actually reverses the dissociation of the enzyme, at least in part, when added after incubation in the absence of substrate (line C, Table IV). The resulting specific activity is higher than that observed in the absence of protamine sulfate (line D), and the polycation, by thus reducing the degree of dissociation, behaves as an acti-
ENZYMES
AT
HIGH
DILUTIONS
37
vator of the diluted enzyme. Earlier experiments with hyaluronidase and P-glucuronidase have led to analogous conclusions (2, 3, 5). It is a well-known fact that polycations and polyanions interact with one another and that each class thus counteracts the biological effects of the other (31). That polyanions might therefore promote the dissociation of dilute enzymes into inact#ive subunits would then appear to be a logical consequence, representing the opposite effect of polycations. Sodium dodecyl sulfat’e, which must be considered to behave as a macromolecular polyanion in aqueous solution because of its micelle formation (32), has indeed been found to cause the dissociation of equine hepatic alcohol dehydrogenase (33). The effect of polyanions on /?-glucuronidase and hyaluronidase has been interpreted as a sequestration of polycations being necessary to prevent dissociation of the enzymes and this phenomenon has been termed activatorcompetitive inhibition (3, 4). Whet’her t’he inhibition of enzymes by sodium dodecyl sulfate or by the other polyanions, described herein and reported in the literat’ure (31), is due to increased dissociation of the enzyme or to other mechanisms cannot be decided unless simultaneous molecular weight determinations of the enzymes at the same enzyme concentrations become available. The presence of strong electronegative groups (sulfate ester or sulfonic groups) appears to be a prerequisite for the inhibition. Nonpolymerit polyanions do not act as inhibitors, but low molecular-weight polymeric polyanions may be powerful enzyme inhibitors, and a tenfold or higher increase in their degree of polymerizat’ion contributed only a slight additional effect. It is noteworthy that the action of polyanions on enzymes is not an unspecific one and that one enzyme may be markedly inhibited by a polyanion of a certain chemical nature, whereas another enzyme is not inhibited by the same polyanion, and vice versa. Factors in addition to multiple anionic groups and macromolecular nature must therefore play an important role in determining the inhibition of enzymes by polyanions.
38
BERNFELD,
BERKELEY,
AND
ACKNOWLEDGMENT We wish to thank Dr. Delbert D. Reynolds, Eastman Kodak Company, Rochester, New York, for providing a sample of polyvinylamine hydrochloride. R,EFERENCES 1. REITHEL, F. J. Advan. Protein Chem. 18, 123 (1963). 2. BERNFELD, P., BERNFELD, H. C., NISSELBAUM, J. S., AND FISHMAN, W. H. J. Am. Chem. Sot. 76, 4872 (1954). 3. BERNFELD, P., JACOBSON, S., AND BERNFELD, H. C. Arch. Biochem. Biophys. 69, 198 (1957). 4. BERNFELD, P., AND TUTTLE, L. P. Federation Proc. 18, 191 (1959). 5. BERNFELD, P., TUTTLE, L. P., AND HUBBARD, R. W. Arch. Biochem. Biophys. 92,232 (1961). 6. BERNFELD, P., DONAHUE, V. M., AND BERKOWITZ, M. E. J. Biol. Chem. 226, 51 (1957). 7. BERNFELD, P., NISSELBAUM, J. S., BERKELEY, B. J., AND HANSON, R. W. J. Biol. Chem. 236, 2852 (1960). 8. BERNFELD, P., AND KELLEY, T. F. J. Biol. Chem. 238, 1236 (1963). 9. KUBOWITZ, F., AND OTT, P. Biochem. 2. 314, 94 (1943). 10. KORNBERG, A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, p. 441. Academic Press, New York (1955). 11. TAYLOR, J. F., GREEN, A. A., AND CORI, G. T. J. Biol. Chem. 173, 591 (1948). 12. TAYLOR, J. F., in “Methods in Enzymology” (S. P. Colowick, and N. 0. Kaplan, eds.), Vol. I, p. 310. Academic Press, New York (1955). 13. FISKE, C. H., AND SUBBAROW, Y. J. Biol. Chem. 66, 375 (1925). 14. NOELTING, G., AND BERNFELD, P. Helv. Chim. Acta 31, 286 (1948). 15. BERNFELD, P., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.),
16. 17. 18. 19.
20. 21. 22. 23. 24. 25.
26. 27.
28. 29. 30. 31.
32. 33.
BIEBER
Vol. 1, p. 149. Academic Press, New York (1955). STELLWAGEN, E., .~ND SCHACHMAN, H. K. Federation Proc. 21, 409 (1962). STELLWAGEN, E., AND SCHACHMAN, H. K. Biochemistry 1, 1056 (1962). DEAL, W. C., AND VAN HOLDE, K. E. Federation Proc. 21, 254 (1962). LEVINTHAL, D., SIGNER, E. R., AND FETHEROLF, K. Proc. Natl. Acad. Sci., U. S. 48, 1230 (1962). FRIEDEN, C., Biochim. Biophys. Acta 27, 431 (1958). FRIEDEN, C., J. Biol. Chem. 234, 809, 815 (1959); 237, 2396 (1962). YIELDING, K. L., AND TOMKINS, G. M. Proc. N&Z. Acad. Sci., U. S. 46, 1483 (1960). FISHER, H. F., CROSS, D. G., AND MCGREGOR, L. L., Nature, 196, 895 (1962). JOHNSON, P., AND MASSEY, V. Biochim. Biophys. Acta 23, 544 (1957). ISEMURA, T., TAKAGI, T., MAEDA, Y., AND IMAI, K. Biochem. Biophys. Res. Commun. 6, 373 (1961). SELA, M., WHITE, F. H., JR., AND ANFINSEN, C. B. Biochim. Biophys. Acta 31,417 (1959). ANRINSEN, C. B., HABER, E., SELA, M., AND WHITE, F. H., JR. Proc. Natl. Acad. Sci., U. S. 47, 1309 (1961). HABER, E., AND ANFINSEN, C. B. J. Biol. Chem. 236, 422 (1961). WHITE, F. H., JR., J. Biol. Chem. 236, 1353 (1961). ANFINSEN, C. B., AND HABER, E. J. Biol. Chem. 236, 1361 (1961). “Metabolic Inhibitors” BERNFELD, P., in (R. M. Hochster and J. H. Quastel, eds.), Vol. 2, p. 437. Academic Press, New York (1963). LUNDGREN, H. P., Textile Res. J. 16, 335 (1945). HERSH, R. T., Biochim. Biophys. Acta 68, 353 (1962).