Acid Residue Peptide Research Articles

Hydrolysis of transforming growth factor-α by cell-surface peptidases in vitro

Human transforming growth factor-alpha (h-TGF alpha), a 50-amino acid residue peptide, was incubated with some purified cell-surface peptidases and with renal microvillar membranes prepared from pig and rat. Hydrolysis was monitored by h.p.l.c. and activity by a biological assay. Prolonged incubation with relatively large amounts of endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A (angiotensin-converting enzyme) caused no observable hydrolysis and no detectable loss of biological activity. Incubation with pig renal microvilli also failed to degrade the peptide. In contrast, rat renal microvilli readily degraded h-TGF alpha, as did endopeptidase-2, which is located in rat renal and intestinal brush borders, but is absent from pig kidneys. This enzyme degraded about 30 nmol of h-TGF alpha/h per mg of protein. The physiological significance of these results is discussed.

Membrane interaction of ‘peptide P’ derived from the repeating motif of properdin

A 24 amino acid residue peptide corresponding to the central part of the ‘thrombospondin-repeat’ motif of the human serum protein properdin was synthesized. The peptide, termed ‘peptide P’, contains three tryptophans near the N-terminus and an arginine cluster close to the C-terminus. Its sequence closely matches a consensus sequence which has been claimed to characterize a sulfatide binding motif. Membrane binding of peptide P was analyzed using changes in it tryptophan emission upon adding small unilamellar vesicles. The peptide bound to the membranes in a way suggesting simple water / membrane partitioning. Analysis of electrostatic effects at different ionic strengths indicated small electrostatic contributions upon interaction with zwitterionic lipid, despite the large charge number ( z = + 4) of the peptide. Membrane affinity was increased by one order of magnitude if the bilayers contained 20% of negatively charged lipid. No difference could be detected whether the charged lipid was sulfatide or phosphatidylglycerol. Strong and rapid vesicle aggregation was evident as the peptide associated with the negatively charged vesicles. In addition, a fluorescent energy transfer assay with vesicles and internal total reflection fluorescence microscopy on supported bilayers were used to study membrane interaction of whole human properdin. No sulfatide specificity could be detected.

Raman spectroscopic studies of model human pulmonary surfactant systems: phospholipid interactions with peptide paradigms for the surfactant protein SP-B

The temperature dependence of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) multilayers, reconstituted with various synthetic peptides for modeling human lung surfactant, was monitored by vibrational Raman spectroscopy. The synthetic peptides consisted, respectively, of residues 59-81 of the human surfactant protein SP-B and 21 amino acid residue peptides containing repeating units of arginine separated by either four or eight leucines (RL4 or RL8). Each peptide demonstrated the ability to reduce significantly the surface tension of analogues of the phospholipid mixture used in the Raman studies. Raman spectroscopic integrated band intensities and relative peak height intensity ratios, two spectral parameters used to determine bilayer disorder, provided sensitive probes for characterizing multilayer perturbations in the reconstituted liposomes. Temperature profiles derived from the various Raman intensity parameters for the 3100-2800-cm-1 carbon-hydrogen (C-H) stretching mode region, a spectral interval representative of acyl chain vibrations, reflected lipid reorganizations due to the bilayer interactions of these peptides. For the three reconstituted multilamellar surfactant systems, the gel-to-liquid-crystalline phase-transition temperatures Tm, defined by acyl chain C-H stretching mode order/disorder parameters, increased from 35 degrees C in the peptide free system to 37-38 degrees C, indicating increased lipid headgroup constraints for the model liposomes. Although the values of Tm were similar for the three recombinant lipid/peptide assemblies, individual phase-transition cooperativities varied significantly between systems and between spectroscopically derived order/disorder parameters.

Crystallization studies of cAMP-dependent protein kinase: Cocrystals of the catalytic subunit with a 20 amino acid residue peptide inhibitor and MgATP diffract to 3.0 Å resolution

Crystallographic studies of the catalytic subunit of cAMP-dependent protein kinase demonstrate that the presence of a 20 amino acid residue peptide inhibitor and MgATP during crystallization yields crystals with a different space group and, more significantly, makes an important difference in the quality of the resulting crystals. Under identical experimental conditions, the kinase crystallizes in a cubic space group P4 132 (a = b = c = 169.24 A ̊ ), when no substrates or inhibitors are present, and in the hexagonal space group P6 122 ( or P6 522) (a = b = 80.16 A ̊ , c = 288.07 A ̊ , α = β = 90 °, γ = 120 °) when a 20-amino acid residue peptide inhibitor and MgATP are present. Moreover, the hexagonal crystal diffracts to a resolution of 3.0 Å, while the cubic crystals diffract to a resolution of 4.0 Å.

Identification of a peptide arising from the specific post-translation processing of secretogranin II

The biological role of secrotogranin II is unknown but it has been suggested that the protein may function as a precursor of one or more biologically active neuroendocrine peptides. We have isolated a 33 amino acid-residue peptide from the brain of the frog Rana ridibunda that shows strong (82%) homology with human presecretogranin II-(182–204)-peptide. This region of secretogranin II has also been very strongly conserved in the rat and bovine proteins. Analysis of the nucleotide sequence of the mammalian secretogranin II cDNAs indicates that the peptide sequence is flanked by two Lys-Arg dibasic residue processing sites. It is proposed, therefore, that this fragment represents a specific product of the post-translational processing of secretogranin II and, by analogy with peptides derived from chromogranin A, may be important in the regulation of neurosecretion.

Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA

DNA from Ehrlich ascites tumor (EAT) cells and from human placenta was examined for covalent bonds between hydroxy amino acid residues in peptides and nucleotide phosphate groups. The residual proteinaceous material in highly purified DNA was radiolabelled with 125Iodine and the linking-groups between peptides and nucleotides released by combined protease and nuclease treatment were investigated with respect to their chemical and enzymatic stabilities. The residual nucleotide(s)-peptide(s) fraction from DNA isolated after prolonged alkaline cell lysis and phenol extraction contains mainly alkali and acid-stable but phosphodiesterase-sensitive peptide-nucleotide complexes which indicates phosphodiesters between tyrosyl residues in peptides and nucleotide phosphates. In contrast, the linking-group fraction from DNA isolated under native conditions contains additional peptide components. (a) Phospho-peptides that co-purify with DNA but that are not covalently bound to nucleotides. (b) A fraction of peptides that is released from nucleotides by alkali in a time and concentration-dependent reaction. Evidence is presented indicating that the latter fraction involves phospho-triesters between hydroxy amino acid residues in peptides and internucleotide phosphates. The phosphodiesters between hydroxy amino acids and nucleotide phosphates representing the predominant class of peptide-nucleotide complexes in alkali-denatured DNA are most likely side products of peptide-nucleotide phospho-triester hydrolysis.

Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicits T cells expressing V beta 8 and V alpha 2-like genes.

Experimental autoimmune uveoretinitis (EAU) is a predominantly T cell-mediated autoimmune disease induced in susceptible animals by active immunization with human or bovine retinal S-Ag or by passive transfer of activated S-Ag or peptide-specific CD4+ T cells. During the course of studies aimed at the identification of T cell and B cell recognition sites in bovine and human S-Ag, a new potent uveitogenic region, located near the carboxy terminus of the molecule, was identified and characterized. Analysis of several synthetic peptides from this region showed that a 14 amino acid residue peptide, BSAg339-352, was highly uveitogenic when injected with adjuvants into Lewis rats. A uveitogenic T cell line, R737, was raised by in vitro selection of lymphocytes from animals immunized with peptide BSAg333-352. Northern blot analysis of mRNA from the R737 T cell line was positive for the rat homologs of murine V beta 8 and V alpha 2 T cell receptor gene probes. Whereas peptide BSAg339-352 defined the pathogenic site, nonpathogenic, proliferative sites were found in close physical association. This region is immediately adjacent to previously characterized pathogenic and proliferative sites contained in residues BSAg352-364. These results, as well as our previous observations, show S-Ag to be a complex molecule with several highly conserved amino acid sequences that can elicit pathogenic T cells with restricted T cell receptor V gene usage capable of active and passive elicitation of experimental autoimmune uveoretinitis.

Structure, function and properties of antibody binding sites

Do antibody combining sites possess general properties that enable them to bind different antigens with varying affinities and to bind novel antigens? Here, we address this question by examining the physical and chemical characteristics most favourable for residues involved in antigen accommodation and binding. Amphipathic amino acids could readily tolerate the change of environment from hydrophilic to hydrophobic that occurs upon antibody-antigen complex formation. Residues that are large and can participate in a wide variety of van der Waals' and electrostatic interactions would permit binding to a range of antigens. Amino acids with flexible side-chains could generate a structurally plastic region, i.e. a binding site possessing the ability to mould itself around the antigen to improve complementarity of the interacting surfaces. Hence, antibodies could bind to an array of novel antigens using a limited set of residues interspersed with more unique residues to which greater binding specificity can be attributed. An individual antibody molecule could thus be cross-reactive and have the capacity to bind structurally similar ligands. The accommodation of variations in antigenic structure by modest combining site flexibility could make an important contribution to immune defence by allowing antibody binding to distinct but closely related pathogens. Tyr and Trp most readily fulfil these catholic physicochemical requirements and thus would be expected to be common in combining sites on theoretical grounds. Experimental support for this comes from three sources, (1) the high frequency of participation by these amino acids in the antigen binding observed in six crystallographically determined antibody-antigen complexes, (2) their frequent occurrence in the putative binding regions of antibodies as determined from structural and sequence data and (3) the potential for movement of their side-chains in known antibody binding sites and model systems. The six bound antigens comprise two small different haptens, non-overlapping regions of the same large protein and a 19 amino acid residue peptide. Out of a total of 85 complementarity determining region positions, only 37 locations (plus 3 framework) are directly involved in antigen interaction. Of these, light chain residue 91 is utilized by all the complexes examined, whilst light chain 32, light chain 96 and heavy chain 33 are employed by five out of the six. The binding sites in known antibody-antigen complexes as well as the postulated combining sites in free Fab fragments show similar characteristics with regard to the types of amino acids present. The possible role of other amino acids is also assessed. Potential implications for the combining regions of class I major histocompatibility molecules and the rational design of molecules are discussed.

Peptide YY: The Ileo-Colonic, Gastric, and Pancreatic Inhibitor

Peptide YY (PYY) was initially isolated from an ex tract of400 kg ofporcine duodenum using a novel chem ical assay that recognizes peptides with amidated car boxyl termini (Tatemoto and Mutt, 1981; Tatemoto, 1982). This latter structural modification is a characteris tic ofmany biologically active brain-gut peptides. Amino acid sequencing of the final purified product demon strated that peptide YY is a 36 amino acid residue pep tide that exhibits structural homology(Floyd et a!., 1976; Tatemoto et a!., 1982) to two other brain-gut peptides: pancreatic polypeptide (PP) and neuropeptide Y (NPY). These three peptides constitute the third family of struc turally related peptides to be isolated from the brain-gut axis. (This family is discussed further in this issue by Price and Greenberg.)

A new family of growth factor-like peptides ‘Trefoil’ disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins)

Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38–39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic ‘trefoil’ disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

Isolation and characterization of bovine pancreastatin

Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248–294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.

Neuropeptide K suppresses feeding in the rat

We studied the effects of neuropeptide K (NPK), a 36 amino acid residue peptide of the tachykinin family, on latency to onset of feeding and cumulative 1 and 2 h food intake in three experimetal paradigms. Intraperitoneal injection of NPK (1.25 and 3.14 nmol) to food-deprived rats delayed the onset of feeding and significantly decreased the cumulative food intake. Intraperitoneal injection of NPK (1.25 and 3.14 nmol) to water-deprived rats produced no effect on subsequent drinking behavior. Similarly, intraperitoneal injection of NPK (3.14 nmol) 15 min before onset of the dark phase (of the light-dark cycle) significantly delayed the occurrence of ingestive behavior and the cumulative food intake was markedly suppressed. Furthermore, administration of NPK intraperitoneally (0.5–3.14 nmol) 15 min before intraventricular (i.c.v.) injection of neuropeptide Y (NPY 0.47 nmol) to satiated rats significantly suppressed NPY-induced feeding and delayed the onset of ingestive behavior. However, when administered centrally prior to NPY injection, NPK delayed the onset of feeding response only. Collectively, these findings show that NPK can acutely and consistently suppress feeding behavior.

Reactivity of consecutive basic amino acid residues in peptides.

Different tetrapeptides of general formula L-Ala-X-X-Gly, possessing a basic doublet in the second and third position (X = Arg or Lys), have been synthesized as free or N-acetylated molecules. The chemical reactivity of the arginine guanidino group and of the lysine epsilon-amino group were studied using respectively the Sakaguchi and the ortho-diacetylbenzene reactions, in the tetrapeptides as well as in related molecules. In both cases, the colour yield is markedly influenced by the length of the polypeptide chain and by the relative positions of the arginine and lysine residues, suggesting the occurrence of intramolecular bonds within the tetrapeptide molecule. Tryptic hydrolysis of the tetrapeptides was followed by evaluating the amino acids or peptides which appear to be specific for the different possible cleavages at the arginyl or at the lysyl bonds. The susceptibility to trypsin of the carboxylic group of the second basic amino acid decreases progressively in the order Lys-Arg greater than Arg-Arg much greater than Lys-Lys greater than Arg-Lys, which shows a fair correlation with the intra-cellular cleavage of the bonds observed during the processing of preproteins of of the precursors of several physiologically active peptides.

Substitution of SER 61 → GLY 61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase

Lipoprotein lipase (LpL) activity is enhanced by apolipoprotein C-II (apoC-II), a 79 amino acid residue peptide. The minimal apoC-II sequence required for activation of LpL resides between residues 56–79. To determine the possible role of an acyl-apoC-II intermediate involving Ser 61 in enzyme catalysis, a synthetic peptide of apoC-II containing residues 56–79 was synthesized and compared to the corresponding peptide with serine at position 61 being substituted with glycine. With two different LpL assay systems, both peptides enhanced enzyme activity. Since glycine does not contain a hydroxyl group, these results rule out the possibility that an acyl-apoC-II intermediate with Ser 61 is required for enzyme activation.

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.

Serine carboxypeptidases. A review

Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes. Most serine carboxypeptidases presumably participate in the intracellular turnover of proteins and some of them, in addition, release amino acids from extracellular proteins and peptides. However, prolyl carboxypeptidase which cleaves the C-terminal peptide bond of angiotensin II and III is a serine carboxypeptidase with a more specific function. Serine carboxypeptidases are usually glycoproteins with subunit molecular weights of 40,000–75,000. Those isolated from fungi apparently contain only a single peptide chain while those isolated from higher plants and animals in most cases contain two peptide chains linked by disulfide bridges. However, a number of the enzymes aggregate forming dimers and oligomers. It is probable that the well-known catalytic mechanism of the serine endopeptidases is also employed by the serine carboxypeptidases but presumably with the difference that the pKa of the catalytically essential histidyl residue is somewhat lower in the carboxypeptidases than in the endopeptidases. However, the leaving group specificity of these two groups of enzymes differ since the carboxypeptidases only release C-terminal amino acids from peptides (peptidase activity) and not longer peptide fragments. In addition, they release C-terminal amino acid amides (peptidyl amino acid amide hydrolase activity) or ammonia (amidase activity) from peptide amides and alcohols from peptide esters (esterase activity) and this property they share with the serine endopeptidases. Like other proteolytic enzymes the serine carboxypeptidases contain binding sites which secure the interaction between enzyme and substrate. In this laboratory, the properties of these have been studied for three serine carboxypeptidases, i.e. carboxypeptidase Y from yeast and malt carboxypeptidases I and II, by means of kinetic studies, chemical modifications of amino acid side-chains located at these binding sites and exchange of such amino acid residues by site-directed mutagenesis. Serine carboxypeptidases, such as carboxypeptidase Y and malt carboxypeptidase II which are available in large quantities, can be applied for several purposes. Their broad specificity and ability to release amino acids from the C-terminus of a peptide chain can be employed in determination of amino acid sequences, and their ability to catalyze transpeptidation reactions and aminolysis of peptide esters can be employed to exchange C-terminal amino acid residues in peptides and in step-wise synthesis of polypeptides, respectively. The type of reactions catalyzed by these enzymes is limited by their specificities but, fortunately, some of the derivatives of carboxypeptidase Y with changed specificity due to chemical modifications and genetic substitutions of amino acid side-chains located at binding sites can be employed with advantage. These modified enzymes are examples on how the different activities of an enzyme can be perturbed by “protein engineering”, hence rendering the enzyme particularly suitable for certain processes.

Enhancement of helper and suppressor T cell activities by thymosin alpha 1 injection in old mice.

A single injection of immunodeficient old (15-24 month old) BDF1 mice with 1-100 micrograms of synthetic thymosin alpha 1 (T alpha 1), a 28-amino acid residue peptide, shortly before horse erythrocyte (HRBC)-priming, enhances the helper activity of their spleen cells. Helper activity of spleen cells from uninjected or T alpha 1-injected HRBC-primed old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from unprimed young (3-month-old) mice and the conjugate 2,4,6-trinitrophenyl (TNP)-HRBC. As evaluated from the in vitro anti-TNP antibody response, spleen cells exhibit higher helper activity when derived from T alpha 1-injected, as compared to uninjected, HRBC-primed old mice. No effect is observed when T alpha 1 is injected in young mice before HRBC-priming. Old mice were also injected with the N14 (N-terminal amino acid residues 1-14) or the C14 (C-terminal amino acid residues 15-28) synthetic fragments of the T alpha 1 molecule. Injection of T alpha 1 or the N14 fragment restores the helper activity of old mice to levels comparable to that displayed by helper cells from young mice. Conversely, injection of the C14 fragment in old mice induces only a negligible increase in helper activity of their spleen cells. T cells from HRBC-primed mice were separated by nylon wool filtration into Th1 (non-adherent) and Th2 (adherent) T cells and these cell subpopulations were assayed for helper activity. Helper activity of Th1 and Th2 cells is found to be impaired by aging when they are tested either separately or upon their recombination.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide K: Isolation, structure and biological activities of a novel brain tachykinin

A 36 amino acid residue peptide, which contains a substance K sequence at its C-terminus has been isolated from porcine brain extracts. The primary structure of the peptide, designated neuropeptide K (NPK), was found to be: Asp-Ala-Asp-Ser-Ser-Ile-Glu-Lys-Gln-Val-Ala-Leu-Leu-Lys-Ala- Leu-Tyr-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2. This N-terminally extended form of substance K is present in a high concentration in the brain. The peptide is highly biologically active with regard to gallbladder contraction, protein extravasation, hypotension and bronchial smooth muscle spasm and may act as an additional tachykinin neuromessenger.

Macroscale high-performance liquid chromatographic separation and instrumental identification of components of diethylaminoethyl murine epidermal growth factor

Murine epidermal growth factor (m-EGF), a polypeptide produced as a chromatographically homogeneous peak on diethylaminoethyl (DEAE) cellulose by the method of Savage and co-workers, and characterised as a single compound, has been shown by ourselves and several other groups to be a mixture. The present contribution extends our previously reported work and discuss the separation of this material, termed DEAE—m-EGF, into its components by preparative ion-pair reversed-phase high-performance liquid chromatography (RP-HPLC) on C 18 μBondapak in quantities up to 50 mg per run. Isocratic elution was used and the mobile phase was acetonitrile—water (26:74, v/v, 0.04 M in triethylamine acetate); pH was 5.6, temperature 40°C, and detection was by ultraviolet absorption at 254 nm, and (for some runs) by differential refractometry. Seven significant peaks, four major, three minor, were detected. Of the major peaks, two designated α- and β-EGF, constituted 70% of the total mass and were the most important to our work. Each of the eluted peaks was recovered by lyophilisation, and this product checked for homogeneity by ion-pair RP-HPLC on a C 18 μBondapak analytical column, with utraviolet detection as before. All recovered peaks were found to be homogeneous by this criterion. These chromatographically homogeneous compounds were investigated by modern physicochemical instrumentation to determine their structure. The molecular weight of each of the species was determined by fast atom bombardment mass spectrometry. High-field proton magnetic resonance at 270 MHz provided structural and conformational information. Polarimetry and ultraviolet absorption were also used to characterise the compound. α-EGF, for example, had a molecular weight of 6040 corresponding to the 53 amino acid residue peptide previously designated EGF; β-EGF had a molecular weight of 5930. This molecular weight differential of 110 suggested the hypothesis that β-EGF was a 52 residue peptide corresponding to α-EGF minus the terminal asparagine at position 1. Proton magnetic resonance difference spectroscopy (β spectrum subtracted from α) provided powerful confirmatory evidence for this hypothesis. All materials recovered from RP-HPLC were tested in the sheep and found to retain their biological activity.

Domains in human plasminogen

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of ε-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.