exist as such in nature or are derived from natural sources. They are treated here as a class distinct from the naturally occurring polypeptide antibiotics and hormones. In general, these inhibitors have not yet been adequately characterized. Except for the basic polypeptide and protein inhibitors, which are thought to act via electrostatic interaction with anionic polymeric sites, little is known of the mechanism of inhibition of this class of substances. The coverage of the voluminous literature pertinent to these inhibitors is representative rather than exhaustive.
A. Viruses as Inhibitors
Virus particles may be considered to be polypeptide inhibitors in the broad sense by virtue of their protein content. The biochemistry, biology, and consequences of viral infection to the cell have been the subjects of several excellent reviews (193-197). Polypeptide structures also play a role in the blocking of hemagglutination (198) and virus adsorption (194, 199, 200), as well as in interference phenomena between viruses (196, 201-203).
Isaacs and Lindenmann (203a) described a soluble factor called inter
feron, which exhibits the properties of a protein and is produced by cells consequent to exposure to infective virus or viral components. These substances appear to be viral inhibitors which enhance cell resistance to virus. The production, biological behavior, and potential significance of the interferons has been reviewed recently (203b).
Β. Antigen-Antibody Phenomena
That many forms of biological antagonism are intimately associated with the periodicity of amino acids and the surface characteristics of poly
peptide compounds is evidenced by the vast range of antigen-antibody phenomena. Sevag (204) considers antigenic proteins to be enzymic, their specific biological activity being the production of specific antibody.
The biological aspects of antibody formation have been the subject of a recent review (205).
The substrate for these reactions is γ-globulin, which, in the presence of antigen, is synthesized in stable configurations determined by the periodicity and surface characteristics of the antigen. Pauling (206) pro
posed that the synthesis of antibody results from a folding of the poly
peptide precursors into a configuration complementary to that of the antigen. The studies of Landsteiner and van der Scheer (207-210) more than thirty years ago with hapten peptides demonstrated that the in
hibition of antigen-antibody reactions represents a direct displacement phenomenon.
More detailed reviews of these topics may be found in Sevag (204),
Martin (211), Landsteiner (212), Burnet (213), and current textbooks on classical immunology and immunochemistry.
C. Enzymes as Inhibitors
1. E N Z Y M E S A S T O X I N S
The classic toxins and endotoxins of many species of bacteria are pro
teins, to which enzyme activity has not yet been attributed (214, 215).
However, bacteria secrete a variety of enzymes, such as hyaluronidases, collagenases, proteases, peptidases, phospholipases, and phosphatases (216), which contribute to the physiological effects of toxins and endotoxins by means of their adverse effects upon the host and thus, in a sense, may be considered to be metabolic inhibitors. The venoms secreted by bees, scorpions, poisonous spiders, snakes, toads, and fish also contain a variety of enzymes which contribute to physiological shock, interfere with blood coagulation, lyse erythrocytes, etc. (216).
There are examples in which enzyme protein appears to function as a toxin. The parallel relationship between the proteolytic and anticoagulant activity of snake venoms is well known (217). Eagle (218) observed a similar relationship between the anticoagulant activity and protease activity of a variety of poisons. Since hematological effects are prominent in snake bite, such proteases must be considered to be toxins. Similarly, those phospholipases which lyse erythrocytes are considered to function as toxins (216).
Among bacterial products, the α-toxin secreted by various species of Clostridium welchii is a lecithinase (phospholipase) {216, 219), the activity of which can be inhibited or blocked by displacement with cerebrosides (220) or antibody (219). The enzymes secreted by other species of the
"gas gangrene" group are antigenically distinct (221), and can be associ
ated only in varying degrees with toxicity, although antibody or substrate appears to be bound at the same locus on the enzyme protein (222).
The role of enzymes as toxins or as components of toxins has been re
viewed more extensively by Zeller (216).
2 . O T H E R I N H I B I T O R Y E N Z Y M E S
The inhibition of enzymes by other enzymes has been reported. Sang -(228) found the inhibitor isolated from the body wall of the round worm, Ascaris lumbricoides, to be a protease. Urease blocks the action of trypsin without losing its own enzymic activity (224).
Lysozyme, a basic mucopolysaccharidase isolated from a wide variety of animal, plant, and bacterial sources, exhibits bacteriolytic activity against both gram-positive and gram-negative bacteria. It is particularly effective against Micrococcus lysodeikticus, B. megaterium, and Sarcina flava. The properties of lysozyme have been reviewed (225). Other basic enzymes reported to possess antibacterial activity are ribonuclease, de-oxyribonuclease, and hyaluronidase. It is possible that the antibacterial properties of these enzymes, active only in high concentrations, were due to the presence of impurities (226). Other inhibitory properties of ribo
nuclease have been summarized by Bergel (226a). Trypsin and chymo-trypsin have been reported to inhibit penicillinase (226b).
Bergel (226a, 227) has reviewed preliminary work on the inhibition of experimental tumors in vivo with milk xanthine oxidase (228), ribonuclease (229), and deoxyribonuclease (229a).
D. Natural Inhibitors of Proteolytic Enzymes
1. N A T U R A L T R Y P S I N I N H I B I T O R S
Trypsin, a proteolytic pancreatic enzyme that hydrolyzes peptide bonds of basic character, is specifically inhibited by several natural proteins which may be involved in the stabilization of various biological fluids
(280-282). These trypsin inhibitors possess molecular weights in the range of 6000-34,000, and have been isolated from pancreas, soybean, lima bean, egg white (ovomucoid), and colostrum. Table I I I , abstracted from Des-nuelle (280), gives some of the properties of the natural trypsin inhibitors.
The inhibition of trypsin seems in most instances to be stoichiometric and
T A B L E I I I
NATURALLY OCCURRING TRYPSIN INHIBITORS
0
Origin
Approximate molecular
weight Isoelectric point
Pancreas I 6500-16,000 8.7-10.0
Pancreas I I 9600 4.5-5.9
Soybean 17,000-24,000 4.5
Lima bean 8000-18,700 < 3 . 6
Egg white (ovomucoid) 9100-34,000 3.8-4.4
Colostrum 10,500 4.2
° Abstracted from P. Desnuelle ref. 280.
reversible. The pancreatic, ovomucoid, and soybean inhibitors are com
petitive. Since the natural inhibitor-trypsin complex is unstable in acid or base and can be destroyed by dilution, the binding evidently is not covalent. By analogy with the inhibition of trypsin by acidic poly-a-amino acids (cf. Section I V , A ) , this binding may be electrostatic in nature.
Trypsin is also inhibited by serum protein (282) and by protein ex
tracts of the body wall of the round worm, A. lumbricoides (223, 233) (cf. Section I I I , C, 2 ) .
2. T H E N A T U R A L P E P S I N I N H I B I T O R
Pepsinogen is the zymogen of pepsin, and must be cleaved by acid or pepsin itself before the proteolytic action of pepsin can be demonstrated (234). The activation of pepsinogen (mol. wt. ca. 43,000) involves the generation of pepsin (mol. wt. ca. 36,000), several neutral peptides (total mol. wt. ca. 4000), and the so-called "pepsin inhibitor" (mol. wt. ca. 3200)
(235). The pepsin inhibitor, a polypeptide with 29 amino acids, appears to be located in the middle of the pepsinogen molecule. Before pepsinogen can be activated, the pepsin-pepsin inhibitor amide bond must be broken.
Figure 1, by Bovey and Yanari (234), is a schematic representation of the location of the pepsin inhibitor in the pepsinogen molecule. Further de
tails are given by Bovey and Yanari (234). This polypeptide inhibits pepsin between pH 5 and 6.
The natural pepsin inhibitor and the synthetic basic poly-a-amino acids, such as polylysine (236) (cf. Section I V , A ) , are effective com
petitive inhibitors of pepsin. Extracts of Ascaris (223, 233) also show antipeptic activity.
FIG. 1. The structure of pepsinogen. The principal attack by pepsin is at points marked P, releasing miscellaneous peptides (A), pepsin inhibitor ( £ ) , and pepsin (C). The undetermined sequences are actually much larger in relation to the known sequences than is indicated. The location of the phosphoserine residue with respect to the di
sulfides is also uncertain. [Reproduced from F . A . Bovey and S. S. Yanari, ref. (234).]
3. N A T U R A L I N H I B I T O R S O F B A C T E R I A L A N D M O L D P R O T E A S E S
Several proteolytic enzymes elaborated by microorganisms are inhibited by naturally occurring protein inhibitors (237). Proteinaceous inhibitors active against B. subtilis alkaline protease, Aspergillus alkaline protease, and trypsin are widely distributed in cereals, beans, and potatoes, but not in fruits or vegetables. Barley, rye, kidney beans, broad beans, and po
tatoes are especially rich in these proteins, which inhibit B. subtilis alka
line protease, Aspergillus alkaline protease, and trypsin, but not Asper
gillus acid protease, pepsin, or papain (238). Soybean trypsin inhibitor and ovomucoid from egg white, which inhibit trypsin (cf. Section I I I , D , 1) do not inhibit B. subtilis or Aspergillus alkaline proteases. The ovoinhibitor component of egg white (289) inhibits all three of these proteolytic en
zymes. Serum contains an inhibitor of Aspergillus protease, which is probably different from serum trypsin inhibitor.
Ε. Antimicrobial Tissue Polypeptides and Proteins
It has long been known that antimicrobial substances can be obtained from various animal sources, notably blood, leucocytes, and lymphatic tissues. Because of incomplete chemical characterization and periodic i
'rediscovery/ ' there is considerable confusion as to the identification of such substances and their biological significance as metabolic inhibitors of infectious processes. Extensive consideration of these substances is beyond the scope of this chapter. The reader is referred to the admirable review of these inhibitors by Skarnes and Watson (226) for further details and references.
1. N A T U R A L A N T I B O D I E S
The substances discussed in this section are complex proteins derived from serum, and are inhibitory to bacteria and to viruses.
a. Complement ("Alexin" "Opsonin"). Complement is a euglobulin-carbohydrate-albumin complex which, in conjunction with normal (non
immune) antibody, accounts for the bactericidal, viricidal, lytic, and phagocytosis-enhancing effects attributable to normal (nonimmune) serum. Although early differences of opinion appeared to have been re
solved and the current consensus is that complement, "alexin," and
"opsonin" represent a single substance (226), Tullis and Surgenor (240) have described two serum proteins which enhance phagocytosis and may be neither complement nor properdin.
b. Normal Antibody. It is generally accepted that, in addition to comple
ment, the antimicrobial effects characteristic of normal (nonimmune) serum require the participation of a second component, the nonspecific normal antibody, as opposed to antibodies elicited following exposure to a specific antigen. The origin and specificity of natural antibody has been the subject of extensive debate (226). Mackie and Finkelstein (241, 242) suggested that normal serum contains specific antibodies which result from natural immunization, whereas Landsteiner (243) suggested the natural existence of a variety of globulins which, by accidental affinity to different antigens, appear to be specific. As pointed out by Skarnes and Watson (226), the problem of the nature of natural antibody un
doubtedly has been confused in many instances by the presence of usually undemonstrable levels of specific antibody in normal (nonimmune) serum.
c. Properdin. Properdin (244) is a euglobulin contained in normal (non
immune) serum, which is concerned with nonspecific resistance to in
fectious agents. Properdin requires the presence of complement and
M g +
+ ions for activity, which is a nonspecific antibacterial and antiviral effect. The biological nature and activity of properdin have been reviewed
(245) >
a n
d Skarnes and Watson (226) have presented the not incon
siderable evidence for the identity of natural antibody and properdin, together with the interrelationships of the various components par
ticipating in the antimicrobial and lytic activities of normal serum. The relationship of properdin and noncellular resistance in general to the problems of neoplastic disease has been reviewed by Southam (246).
2. B A S I C P O L Y P E P T I D E S A N D P R O T E I N S
Most of the polypeptide and protein inhibitors of tissue origin are basic in nature, and have been derived principally from cellular elements.
These substances generally are most active against gram-positive bacteria.
Their inhibitory effects are probably the result of electrostatic interaction with negatively charged bacteria or viruses, and the mechanism of action is presumably similar to that of the synthetic basic polypeptides discussed in Section IV.
a. Nucleins, Historiés, and Protamines. The nucleins, complexes of nucleic acids with simple basic proteins such as histones or protamines, are active against gram-positive bacteria (226).
The histones contain large amounts of the two basic amino acids, lysine and arginine, whereas the protamines are rich in arginine and usually low in lysine content. The antibacterial properties of the histones and the protamines have been reported (226, 247-249). The antibacterial activity of a calf thymus histone containing a large amount of lysine has been described (250). Other antibacterial histones have been derived from the basic tissue polypeptides discussed below.
Protamines from various sources have been shown to inhibit bacteria, viruses, a trypanosome, and a yeast. Clupeine, a protamine, has been reported to inactivate bacteriophage (251). Clupeine sulfate suppresses the activity of vaccinia virus (252). Both vaccinia virus and bacteriophage have negatively charged surfaces, allowing combination with basic sub
stances (253).
Skarnes and Watson (254) isolated leukin, an arginine-rich protamine with activity against gram-positive bacteria, from rabbit polymorpho
nuclear leucocytes. Leukin or leukin-like substances have been obtained from the leucocytes of several species, and are presumably protamine or histone fractions of nucleoprotein origin (226).
b. Basic Tissue Polypeptides. Bloom et al. (255) reported the inhibition of B. anthracis by a basic polypeptide, containing a large proportion of
lysine, which was isolated from several animal tissues. Similar basic tissue polypeptides are active against a number of other bacteria (266, 257) and against a bacteriophage (258). Watson and Bloom (258) have correlated antibacterial activity with the high lysine content of the molecule. By fractionation of histone preparations, Crampton, Moore, and Stein (259, 260) obtained a lysine-rich fraction (histone A ) and an arginine-rich frac
tion (histone B). Skarnes and Watson (261) showed the tissue peptide of Bloom et al. (255) to be identical with histone A (259), which is most active at alkaline pH.
A basic polypeptide from calf thymus containing about 40% of lysine and arginine by weight was found to exhibit activity against M. tuber
culosis (262, 268). Hirsch (264) reported that under certain in vitro condi
tions, the arginine-rich histone Β (259, 260) exerted bactericidal activity against various coliform bacilli and micrococci, but that the lysine-rich histone A (259, 260) manifested no significant antibacterial action.
3. M I S C E L L A N E O U S P R O T E I N S W I T H A N T I B A C T E R I A L A C T I V I T Y
The proteinaceous inhibitors discussed in this section are difficult to classify because of even less certain structure, purity, and mechanism of action. Although these substances are discussed in the singular form, each one is probably a mixture and undoubtedly varies in kind according to the source and method of isolation. Except for phagocytin, these sub
stances are all effective against gram-positive bacteria. With the excep
tion of substance M and the antistaphylococcal serum factor, this group of inhibitors has been adequately reviewed by Skarnes and Watson (226).
Phagocytin, a bactericidal substance obtained from rabbits, and limited in distribution mainly to the polymorphonuclear leucocyte, appears to be a protein with general properties characteristic of a globulin. It is different from properdin and lysozyme (265, 266).
β-Lysin is a poorly characterized bactericidal substance isolated from human, horse, and dog serum. It is presumably a protein.
Plakin, an antibacterial material from the blood platelets of the horse, is thought to be a protein and may be related to leukin.
Lactenin is a bactericidal protein found in the whey of human, cow, and goat milk.
Mascherpa (267) has described the isolation of substance M from the organs of tuberculosis-free mammals. This product, a mixture containing a polypeptide component, is reported to exhibit activity against M. tuber
culosis in vitro and in vivo.
Yotis and Ekstedt (268, 269) have described a partially purified, heat-stable, antistaphylococcal serum factor, which occurs primarily in a
water-soluble globulin fraction of normal human, rabbit, and horse serum. It has not yet been demonstrated in bovine serum. This antibacterial factor, which appears to be distinct from lysozyme, exerts a nonspecific (partially lytic), lethal effect upon several species of gram-positive bacteria. Anti
bacterial activity, except that exhibited against M. lysodeikticus, can be blocked by exposure of the cells to staphylococcus coagulase (268, 269).
This factor has been shown to interfere with the oxidation of glucose (269), but otherwise its mechanism of action is unknown.
There are a number of protein-like compounds known as bacteriocins and bacteriocin-like substances elaborated by several bacterial species which are inhibitory for the cells which produced them, i.e., their bio
synthesis by the cell is self-lethal. Some of these substances exhibit in
hibitory activity against a limited spectrum of related bacterial species.
The reader is referred to the recent review by Ivancvics (269a) for further details.
As pointed out recently by Nigrelli (269b), it is now well established that many marine organisms produce promoting and growth-inhibiting substances, including antibiotics in the classic sense. Although information concerning the biochemical nature and mechanism of action of such substances in well-defined biological systems is incomplete, certain of these substances will undoubtedly prove to be peptides or proteins. Li et al. (269c), for example, have described a class of compounds designated as paolins which have been isolated from abalone and oysters. These substances are nondialyzable, appear to be proteins (probably muco-proteins), and exhibit antiviral as well as antibacterial activity.
F. Inhibitory Properties of Protamines
The cationic nature, basic amino acid content, and antimicrobial proper
ties of the protamines have been discussed (cf. Section I I I , E, 2) and have been reviewed elsewhere (226, 247, 248). In this section, other inhibitory properties of the protamines are described.
1. H E P A R I N A N T A G O N I S M
Protamine, a basic protein, and heparin, an acidic polysaccharide, are both anticoagulants. However, the clotting time of heparinized blood can be restored to normal by the inhibition of heparin with protamine (270).
This stoichiometric reaction of protamine with heparin is the basis for the so-called heparin-protamine titration. The inhibition is presumably through electrostatic forces. Similar antiheparin effects have been observed with synthetic basic polypeptides (cf. Section IV, B ) .
2. E N Z Y M E I N H I B I T I O N
Muscle phosphorylase a is effectively inhibited by the protamine, salmine (271). Madsen and Cori (272) have proposed a mechanism of inhibition in which an insoluble enzyme-inhibitor complex is formed by the op
positely charged enzyme and protamine. Protamine also inhibits lipo
protein lipase (clearing factor), an enzyme present in postheparin plasma and in normal rat hearts (278).