Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Biological activity of a fluorescein human growth hormone derivative prepared by specific covalent labeling of lysine-70

Modification of human growth hormone (hGH) with a low equimolar concentration of fluorescein isothiocyanate (FITC) yielded a derivative containing 1 mol of fluorescein/mol of protein. The site of modification was identified as lysine-70. Lysine-70 of hGH is about 3-fold more reactive than a "normal" lysine in a protein, having pseudo-first-order kinetics Kobs = 110 +/- 7 M-1 min-1 at pH 10.5. The pKa of the lysine was estimated to be 10.7, within the normal range of normal epsilon-lysine moieties in proteins. This higher chemical reactivity seems to favor selective labeling of this moiety at low FITC concentrations. To obtain monomodified derivatives, hGH was derivatized with 0.6 equiv of FITC, and the modified derivatives were separated from unreacted hormone by means of HPLC using a Mono Q column. Its biological activity, determined by Nb2 bioassay, decreased to 40%, and its affinity toward lactogen receptors in Nb2 cells and toward somatogen receptors in bovine liver decreased respectively to 30% and 20%. The present study indicates that out of the seven amino groups of human growth hormone, the epsilon-amino group of lysine-70 is excessively reactive toward FITC. Second, this particular amino group contributes to receptor binding and receptor activation. Lysine-70 is located in the loop between the first and second helix and close to the carboxy-terminal end of the first helix. This contribution is most likely the result of the formation of an electrostatic interaction between the hormone and the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B

The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl-l-alanylglycyl-l-phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl-l-leucyl-l-phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S₁ and S₂, on the surface of SGPB which accommodate the P₁ and P₂ side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the N^?2 atom and O^γ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (K_I) allows one to estimate the binding energy of the leucyl side-chain as ?2.6 kcal mol^?1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.     

162 Dominant forces in protein folding

In the first part of this talk, I will discuss the need for a paradigm shift from hydrophobic (HφO) to a hydrophilic ((HφI) based theory of protein folding. Next, I will discuss the various types of solvent-induced forces that are exerted on various groups on the protein. It is argued, both theoretically and by simulations, that the HφI–HφI solvent-induced forces are likely to be the strongest. Therefore, it is suggested that these forces are also the forces that force the protein to fold, in a short time, along a narrow range of pathways. This paradigm shift also answers Levinthal’s question about the factors that “speed” and “guide” the folding of proteins.     

Nuclear accumulation of multiple protein kinases during prolactin-induced proliferation of Nb2 rat lymphoma cells

Intracellular kinases play important roles in signal transduction and are involved in the surface receptor-mediated regulation of cellular functions, including mitogenesis. In the present study, we examined the possible involvement of various protein kinases in the passage of a mitogenic signal from the cell surface to the nucleus of Nb₂ cells, a rat nodal lymphoma cell line in which prolactin is a mitogen. Following a prolactin challenge, various kinase activities were monitored at short intervals in different cellular fractions over a 60 min period. Protein kinase C (PKC) activity in the cytosolic fraction rapidly declined to 50% of its original activity within the first 30 min, while PKC activity in the nuclear fractions increased sharply, reaching its highest level by 30 min following a prolactin challenge. There were also increases in both casein kinase and protein tyrosine kinase (PTK) activities in the nuclear fractions during the first 30 min following a prolactin challenge that paralleled PKC activity. The activities of all three kinases declined thereafter, reaching levels close to their respective basal values by 60 min following initiation of prolactin treatment. These observations suggest the possibility that multiple protein kinases may be involved in mitogenic signal transduction for prolactin in Nb₂ cells. © 1996 Wiley-Liss, Inc.     

Comparative mitogenic and galactopoietic effects of IGF-I, IGF-II and Des-3-IGF-I in bovine mammary gland in vitro.

Insulin-like growth factors (IGFs) I and II (IGF-I, IGF-II) and Des-3-IGF-I at physiological concentrations are potent mitogens of bovine undifferentiated mammary epithelial cells cultured in collagen in a serum-free medium. Des-3-IGF-I was found to be as potent as IGF-I, while IGF-II was significantly less active. All three factors acted either synergistically or additively with epidermal growth factor (EGF), cholera toxin and fetal calf serum (FCS). Indirect evidence indicates that despite its lower mitogenic activity the action of IGF-II is mediated through IGF-I receptors. The galactopoietic activity of Des-3-IGF-I and IGF-II was studied in an organ culture of bovine lactating mammary glands using lactogen-responsive fat synthesis as a test. Neither Des-3-IGF-I nor IGF-II exhibited galactopoietic activity nor did they affect the galactopoietic activity of prolactin.     

The effects of external compression on venous blood flow and tissue deformation in the lower leg

External pneumatic compression of the lower legs is effective as prophylaxis against deep vein thrombosis. In a typical application, inflatable cuffs are wrapped around the patient's legs and periodically inflated to prevent stasis, accelerate venous blood flow, and enhance fibrinolysis. The purpose of this study was to examine the stress distribution within the tissues, and the corresponding venous blood flow and intravascular shear stress with different external compression modalities. A two-dimensional finite element analysis (FEA) was used to determine venous collapse as a function of internal (venous) pressure and the magnitude and spatial distribution of external (surface) pressure. Using the one-dimensional equations governing flow in a collapsible tube and the relations for venous collapse from the FEA, blood flow resulting from external compression was simulated. Tests were conducted to compare circumferentially symmetric (C) and asymmetric (A) compression and to examine distributions of pressure along the limb. Results show that A compression produces greater vessel collapse and generates larger blood flow velocities and shear stresses than C compression. The differences between axially uniform and graded-sequential compression are less marked than previously found, with uniform compression providing slightly greater peak flow velocities and shear stresses. The major advantage of graded-sequential compression is found at midcalf. Strains at the lumenal border are approximately 20 percent at an external pressure of 50 mmHg (6650 Pa) with all compression modalities.     

Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1

Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. We found that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation was dependent upon Ena/VASP function and directly correlated with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function was required for filopodial formation from the growth cone in response to global PKA activation. We propose that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues.     

Exploring the origin of the ion selectivity of the KcsA potassium channel

The availability of structural information about biological ion channels provides an opportunity to gain a detailed understanding of the control of ion selectivity by biological systems. However, accomplishing this task by computer simulation approaches is very challenging. First, although the activation barriers for ion transport can be evaluated by microscopic simulations, it is hard to obtain accurate results by such approaches. Second, the selectivity is related to the actual ion current and not directly to the individual activation barriers. Thus, it is essential to simulate the ion currents and this cannot be accomplished at present by microscopic MD approaches. In order to address this challenge, we developed and refined an approach capable of evaluating ion current while still reflecting the realistic features of the given channel. Our method involves generation of semimacroscopic free energy surfaces for the channel/ions system and Brownian dynamics (BD) simulations of the corresponding ion current. In contrast to most alternative macroscopic models, our approach is able to reproduce the difference between the free energy surfaces of different ions and thus to address the selectivity problem. Our method is used in a study of the selectivity of the KcsA channel toward the K+ and Na+ ions. The BD simulations with the calculated free energy profiles produce an appreciable selectivity. To the best of our knowledge, this is the first time that the trend in the selectivity in the ion current is produced by a computer simulation approach. Nevertheless, the calculated selectivity is still smaller than its experimental estimate. Recognizing that the calculated profiles are not perfect, we examine how changes in these profiles can account for the observed selectivity. It is found that the origin of the selectivity is more complex than generally assumed. The observed selectivity can be reproduced by increasing the barrier at the exit and the entrance of the selectivity filter, but the necessary changes in the barrier approach the limit of the error in the PDLD/S-LRA calculations. Other options that can increase the selectivity are also considered, including the difference between the Na+...Na+ and K+...K+ interaction. However, this interesting effect does not appear to lead to a major difference in selectivity since the Na+ ions at the limit of strong interaction tend to move in a less concerted way than the K+ ions. Changes in the relative binding energies at the different binding sites are also not so effective in changing the selectivity. Finally, it is pointed out that using the calculated profiles as a starting point and forcing the model to satisfy different experimentally based constraints, should eventually provide more detailed understanding of the different complex factors involved in ion selectivity of biological channels.