Functional independence of the kringle 4 and kringle 5 regions of human plasminogen.
As part of continuing studies to evaluate whether the kringle domain regions of human plasminogen (HPg) exhibit independent conformational properties, simple model systems are required. Toward this end, we have constructed cDNA regions of HPg encoding its kringle 4 ([K4HPg]) and kringle 4-5 ([K4HPgK5HPg]) regions, expressed these gene fragments in bacterial cells, and purified the recombinant (r) products. The resulting r-[K4HPgK5HPg] was also employed to obtain the r-[K5HPg] domain of HPg by limited elastolytic digestion of this double-kringle polypeptide. The omega-amino acid ligand binding properties and thermal denaturation characteristics of r-[K4HPg], r-[K5HPg], and r-[K4HPgK5HPg] were determined, along with those for the [K5HPg] domain linked to the protease (P) region of HPg ([K5HPg]P). This allowed us to evaluate whether the conformational properties of the [K5HPg] module were influenced by the presence of its neighboring domains in HPg. The temperature midpoint of maximum heat capacity, Tm (and calorimetric enthalpy, delta H), for thermal denaturation of r-[K4HPg] was 57.8 degrees C (79.8 kcal/mol) in the absence of epsilon-aminocaproic acid (EACA) and 70.8 degrees C (93.7 kcal/mol) in the presence of that ligand. The corresponding values for isolated r-[K5HPg] were 50.4 degrees C (78.4 kcal/mol) and 61.0 degrees C (89.8 kcal/mol), respectively. These parameters for the isolated kringles were essentially unchanged when these same kringle domains were present in the r-[K4HPgK5HPg] and [K5HPg]P covalently linked pairs. Similarly, the thermodynamic characteristics (delta G, delta H, and delta S) that describe the binding energy of EACA to r-[K4HPg] at 25 degrees C were -6.3 kcal/mol, -4.5 kcal/mol, and 6.0 eu, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
N
N
Menhart
S G
SG
McCance
L C
LC
Sehl
F J
FJ
Castellino
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004247:DNA; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D010958:Plasminogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D000596:Amino Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA