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Publisher Summary This chapter focuses on the quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography (HPLC). It presents several methods in which the HPLC analysis of fibrinopeptides can yield (1) initial rates of thrombin-catalyzed release of fibrinopeptides from fibrinogen, (2) the Miehaelis–Menten parameters catalytic constant( k CAT ) and (Michaelis constant) K M for a-thrombin-catalyzed release of fibrinopeptide A (FPA) and fibilnopeptide B (FPB) from fibrinogen and fibrin I, (3) the specificity constant k CAT / K M for both FPA and FPB at concentrations of fibrinogen below K M , and (4) the inhibition constant for hirugen, an exosite inhibitor of α-thrombin. The chapter also discusses the HPLC profile of coeluting fibrinopeptides and activation peptides from factor XIII. The chapter describes a method for determining the concentration of α-thrombin that is catalytically competent to process fibrinogen.

Human and bovine α-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent ζ-thrombins, which are comprised of two noncovalently linked fragments: a 36- (human) or 49- (bovine) residue A-chain linked by a disulfide to B-chain residues B1–148 (ζ1-thrombin) and B-chain residues B149–259 (ζ2-thrombin). Human and bovine d-Phe-Pro-Arg-CH2-ζ- and PhMeSO2-ζ-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine d-Phe-Pro-Arg-CH2-ζ- and PhMeSO2-ζ-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine ζ1-thrombin:human ζ2-thrombin and human ζ1-thrombin:bovine ζ2-thrombin. Human ζ1-thrombin and ζ2-thrombin were isolated, and, upon recom-bining, the isolated fragments refolded to generate catalytically competent ζ-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native α-thrombin. Both α- and ζ-thrombin refolded via first-order processes (k = 0.011–0.014 s−1), which in the case of ζ-thrombin were independent of whether ζ1-thrombin and/or ζ2-thrombin was incubated in refolding buffer prior to mixing. This observation, together with others, is consistent with the view that generation of catalytically competent enzyme proceeds via a kinetic pathway wherein ζ1-thrombin and ζ2-thrombin independently partially refold and form a noncovalent complex that undergoes a rate-determining rearrangement to active thrombin.

Highly purified GH-receptor preparations from 3T3-F442A fibroblasts, whose differentiation into adipocytes is promoted by GH, have been shown to contain a tyrosine kinase capable of phosphorylating GH receptors. In the current work, characteristics of the tyrosine kinase responsible for the in vitro phosphorylation of GH receptors from cultured 3T3-F442A fibroblasts were examined, and the presence of this GH receptor-associated tyrosine kinase activity was demonstrated in multiple cell types. GH-receptor complexes from GH-treated cells were partially purified by immunoprecipitation using anti-GH antibodies and then incubated as an immune complex with [gamma 32P] ATP. Incorporation of 32P into the GH receptor from 3T3-F442A fibroblasts was apparent within 1 min at 30 C after the addition of [gamma 32P]ATP (5-10 microM). A divalent cation was requisite for the phosphorylation; Mn2+ was significantly more effective than Mg2+ and Co2+; Ba2+, Ca2+, or Zn2+ had no effect. Excess unlabeled ATP, but not cytosine triphosphate, GTP, or uridine triphosphate, abolished 32P incorporation into the GH receptor and [gamma 32P]GTP could not replace [gamma 32P]ATP as a source of 32P. At 5.5 mM Mn2+, phosphorylation exhibited a biphasic dose response to ATP, with maximal phosphorylation occurring at a concentration of 10 microM ATP. At more physiological concentrations of ATP (1 mM), phosphorylation of the GH receptor was also stimulated by lower concentrations of Mn2+ (as low as 500 nM). Optimal reaction conditions determined for the phosphorylation reaction in 3T3-F442A fibroblasts were used to demonstrate incorporation of 32P from [gamma 32P]ATP into partially purified GH receptors from cultured human IM-9 lymphocytes, murine 3T3-F442A adipocytes, rat H-35 hepatoma cells, and freshly isolated rat adipocytes. The 32P was shown to be incorporated into tyrosyl residues in receptors from the two cell types tested (IM-9 lymphocytes and rat adipocytes). Cross-linked [125I] hGH-receptor complexes solubilized from the four cell types (IM-9 lymphocytes, 3T3-F442A adipocytes, H-35 hepatoma cells, and freshly isolated rat adipocytes) bound to and could be eluted from phosphotyrosyl antibody, suggesting that tyrosyl phosphorylation of GH receptors in all of these cells occurs in vivo. The presence of tyrosine kinase activity associated with GH receptors in multiple cell types from different species is consistent with tyrosine kinase activity playing a role in the actions of GH.

Binding of the chromogenic ligand p -nitrophenyl α- d -mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant ( k a ) for complex formation ( k a = 54,000 s −1 m − at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μ m in combining sites and in excess of the ligand. The apparent first-order rate constant ( k −a ) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α- d -mannopyranoside ( k −a = 6.2 s −1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio k a − a (0.9 × 10 4 m −1 ) was in reasonable agreement with value of 1.1 ± 0.1 × 10 4 m −1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln( k a T ) and ln( k a T ) vs 1 T were linear ( T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.

A method is presented for purifying papain from extracts of papaya latex. The procedure involves precipitation of the extract of papaya latex with sodium chloride followed by affinity chromatography of the redissolved precipitate. Precipitation of the protein from the latex extract is necessary to separate the papain from material which interferes with the binding of papain to the affinity column. During affinity chromatography, the affinity column is overloaded to insure absence in the final product of impurities which are capable of binding to the affinity column. The papain prepared by this procedure yielded an amino acid analysis and an N-terminal amino acid analysis expected for a sample of pure papain. No Met was detected on amino acid analysis nor was the presence of N-terminal residues other than He detected. On polyacrylamide disc gel electrophoresis at pH 4.3, papain prepared by the method described in this work was indistinguishable from crystalline papain which was prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Both disc gel patterns consisted of a single band and a trailing shadow which was less than 5% of the main band. In routine spectrophotometric assays, the specific activity toward N,α-benzoyl- l -arginine ethyl ester of papain prepared by the procedure described in this work was indistinguishable from crystalline papain prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Values of 24 sec−1' and 15 m m were obtained from the turnover number and Km for the papain-catalyzed hydrolysis of N,α-benzoyl- l -arginine ethyl ester at 25 °C, pH 6.00, Γ 2 0.30 using a pH stat.

The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.

Steady state kinetic parameters were evaluated for the hydrolytic release of fibrinopeptides A and B (FPA and FPB) from human fibrinogen by human thrombin at pH 7.4, 37 degrees C, and gamma/2 0.15. At low concentrations of fibrinogen (less than 0.4 microM), the release of FPA from A alpha-chains was first order with respect to both the concentration of fibrinogen A alpha-chains and thrombin. The second order rate constant yielded a value of 11.6 (+/- 0.3) X 10(6) M-1 S-1 for the specificity constant (kcat/Km) for this process. Values of 84 (+/- 4) S-1 and 7.2 (+/- 0.9) microM were evaluated for kcat and Km for the thrombin-catalyzed release of FPA from normal human fibrinogen. The amino acid replacement ArgA alpha 16 leads to His present in fibrinogen Petoskey was shown to result in a 160-fold decrease in the specificity constant for hydrolysis at A alpha 16 and concomitant release of FPA. A kinetic analysis for determination of the sequentiality of release of fibrinopeptides was presented. It indicated that at least 97% of FPB was released after FPA. The specificity constant for release of FPB from intact fibrinogen (if it occurs) was less than 3% of that for release of FPA and less than 10% of that for release of FPB from des-A fibrinogen. The specificity constant for the release of FPB from des-A fibrinogen was 4.2 (+/- 0.2) X 10(6) M-1 S-1. The polymerization inhibitors EDTA and Gly-Pro-Arg-Pro inhibited release of FPB but not FPA. These observations are consistent with the generally accepted view that the predominant pathway for the conversion of normal human fibrinogen to fibrin is one wherein FPA is released, des-A fibrinogen polymerizes, and then FPB is released.

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.

Measurements of the efficiency of singlet-singlet energy transfer were used to determine the distance between the hydrophobic binding site and the thiol group required for carbohydrate-binding activity of lima bean lectin. 1-Anilino-8-naphthalenesulfonate, bound to the hydrophobic binding site by noncovalent interactions, was used as the donor. Two different nonfluorescent probes were used as the acceptors: a mercurial, 2-chloromercuri-4-nitrophenol, and a maleimide, 4-dimethylaminophenylazophenyl-4'-maleimide. Acceptor was covalently attached to the thiol group at the putative carbohydrate binding site. The efficiency of energy transfer in both the 1-anilino-8-naphthalenesulfonate/2-chloromercuri-4-nitrophenol and and 1-anilino-8-naphthalenesulfonate/4-dimethylaminophenylazophenyl-4' -maleimide donor-acceptor systems indicated an apparent distance of 28 A between the two sites, assuming that the transition dipole of the donor is not correlated with respect to that of the acceptor and that each donor is quenched by a single acceptor. Using an alternate model wherein each donor is equally quenched by two acceptors on adjacent subunits, an apparent distance of 33.4 A was calculated. The fact that two donor-acceptor pairs with different Forster's critical distance parameters yielded the same distance between the sites is consistent with our assumption of uncorrelated donor-acceptor transition dipoles.

We have employed site-directed mutagenesis to investigate the contribution of a conserved arginyl residue to the catalytic activity and cofactor affinity of D-serine dehydratase, a model pyridoxal 5'-phosphate (vitamin B6) enzyme. Replacement of R-120 in the active site peptide of D-serine dehydratase by L decreased the affinity of the enzyme for pyridoxal 5'-phosphate by 20-fold and reduced turnover by 5-8-fold. kappa cat displayed modified substrate alpha-deuterium isotope effects and altered dependence on both temperature and pH. Analysis of the pH rate profiles of DSD and the R-120----L variant indicated that R-120 interacts electrostatically with catalytically essential ionizable groups at the active site of wild type D-serine dehydratase. The decrease in cofactor affinity observed for DSD(R120L) was not accompanied by significant perturbations in the UV, CD, or 31P NMR spectrum of the holoenzyme, suggesting that the contribution of R-120 to pyridoxal phosphate affinity may be indirect or else involve an interaction with a cofactor functional group other than the 5'-phosphoryl moiety. The properties of two other site-directed variants of D-serine dehydratase indicated that the pyridoxal 5'-phosphate:K-118 Schiff base was indifferent to a small change in the shape of the side chain at position 117 (I-117----L), whereas replacement of K-118 by H resulted in undetectable levels of enzyme. A poor ability to bind cofactor may have rendered DSD(K118H) susceptible to intracellular proteolysis.

Formation of stable blood clots requires formation of glutamyl-6-lysyl peptide crosslinks between certain glutamyl and lysyl residues in fibrin. Formation of these crosslinks is catalyzed by the transamidase factor XIIL. Production of this enzyme is initiated by thrombin-catalyzed limited proteolysis of the inactive zymogen factor XI11 during the final stage of the blood clotting cascade.' This initial proteolytic event in the reaction pathway for conversion of factor XI11 to factor XIIL involves cleavage at Arg-a36 and release of a 36 amino acid residue activation peptide from the a chains of the qbz molecules comprising plasma factor XIII? Using an HPLC procedure developed to monitor the release of peptides upon treatment of human factor XI11 with human a-thrombin, two peptides, AP and AP', with amino acid compositions consistent with that reported2 for activation peptide (TABLE l), were produced upon treatment of factor XI11 with thrombin? The appearance in lower amounts (< 20%) of the second activation peptide (AP') may reflect the presence of a factor XI11 variant in the pooled plasma from which the protein was purified, or it could reflect partial hydrolysis at a Gln or Asn residue during purification. Treatment of factor XIII, or isolated AP and AP', with alkaline phosphatase had no effect on the relative amounts or elution positions of AP and A P , suggesting that activation peptide does not exist in different states of phosphorylation as does fibrinopeptide A (FPA). Incubation of acidified mixtures of AP and AP' for several hours prior to HPLC resulted in the appearance of a shoulder on both the AP and AP' peaks, but no interconversion of the AP and AP'. The appearance of the shoulder may well reflect an N-0 acyl shift in the N-acetylseryl residue at the amino terminus of the activation peptide. Additional studies are required, however, to determine the structural basis for the different elution positions of AP and AP' and for the acid catalyzed alterations of these peptides. All kinetic parameters reported in this work are for the release of the major activation peptide, AP.

Schiff base formation, transaldimination, and reaction with aminothiols are important reactions which occur on the surface of pyridoxal-P-requiring enzymes. As a first step in assessing the role of the protein in these reactions, models for these reactions were studied in the absence of enzyme at 25 degrees, gamma/2 0.28. The reaction of 6-aminocaproic acid with pyridoxal-P to form the Schiff base N6-(P-pyridoxylidene)-aminocaproic acid was studied as a model for formation of Schiff base on the holoenzyme...

Kinetics of binding of p-nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (M2) to concanaviln A (con A) were examined. The time course of formation of a M2 . con A complex is clearly biphasic, whereas the association with con A of p-nitrophenyl 2-O-methyl-alpha-D-mannopyranoside and other monosaccharides is a monophasic process. The biphasic time course of the binding of M2 to conA is most simply explained in terms of a model wherein the disaccharide can bind to con A two different ways. In the initial rapid phase of the biphasic reaction, both complexes form in amounts determined by the relative values of the rate constants for association. In the subsequent slow phase, the complexes equilibriate according to the relative values of the initial constants for formation of each complex. The enthalpy of activation for formation of the initial complexes with M2 is about 4 kcal/mol less favorable than for monosaccharides, whereas the entropy of activations about 14 e.u. more favorable for binding of the disaccharide. These differences in the activation parameters for binding M2 and monosaccharides suggest that con A interacts simultaneously with groups on both mannopyranosyl residues.

A general method is presented for analysis of the resolution of pyridoxal-P-requiring enzymes by carbonyl reagents. The method is useful for accurately determining the very small equilibrium constants (KP) which characterize the dissociation of cofactor from many pyridoxal-P-requiring enzymes. The analysis also establishes the minimum number and relative stabilities of distinct enzymic species involved in the resolution process. Analysis of the resolution of D-serine dehydratase by L-and D-cysteine resulted in the establishment of an enzyme bound thiazolidine derivative as an intermediate in the pathway for resolution. The over-all equilibrium constant (KR) for the reaction, D-serine dehydratase + cystein in equilibrium KR thiazolidine derivative +D-serine apodehydratase was determined. At pH 7.80, T/2 0.33, 25 degrees, KR equal to 1.08 times 10-minus 3. A value of 7.0 nM for the equilibrium constant for the dissociation of D-serine dehydratase to apoenzyme and free pyridoxal-P was determined from the ratio KR/KT, where KT is the equilibrium constant for the formation of a thiazolidine derivative from free pyridoxal-P and cysteine. An estimate of 14 nM for KP was also obtained from partial resolution of D-serine dehydratase by high dilution. The difficulties associated with this direct determination of KP from the dependence on the enzyme concentration of the activity of very dilute solutions of enzyme are discussed.

An immobilized amino acid napbthylamide substrate for proteinases with tryptic-like specificity was prepared by reacting L-arginine P-naphthylamide with an N-hydroxysuccinimide-activated derivative of agarose. Hydrolysis of the immobilized substrate (AlO-Arg-PNA) was followed by monitoring the increase in fluorescence accompanying the release of /3-naphthylamine. Assays using AIO-Arg-PNA were designed for quantitatively determining the presence of l-2 pmol of trypsin and 15 pmol of thrombin. Profibrinolysin, fibrinolysin, and urokinase have either no or very low activities with AlO-Arg-PNA. Trypsin complexed with a,-macroglobulin has no activity (~0.5%) toward AlO-Arg-PNA. The use of AlO-Arg-PNA for monitoring trypsin-a,-macroglobulin titrations was demonstrated. Soluble naphthylamide derivatives of amino acids and peptides have been used widely as substrates in assays of proteolytic enzymes (e.g., l-3), since the P-naphthylamine released on hydrolysis can be determined fluorometrically or calorimetrically, after a diazocoupling reaction. In this study, we describe the preparation and use of a naphthylamide substrate (AlO-Arg+NA) linked to a solid support. Well-defined chromoNH2

31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli d -serine dehydratase, a model pyridoxal 5′-phosphate requiring enzyme that converts the growth inhibitor d -serine to pyruvate and ammonia. Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 °C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+. Na+ antagonized the K+ promoted appearance of the second resonance. These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to OPO bond angle, conformation, and/or hydrogen bonding of the phosphate group. An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca. 10 m m for the equilibrium constant for dissociation of K+ from d -serine dehydratase. The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s−1 at pH 7.8, 25 °C.

p -Nitrophenyl 2- O -α- d -galactopyranosyl-α- d -mannopyranoside and p -nitrophenyl 2- O -α- d -glucopyranosyl-α- d -mannopyranoside were synthesized and the interactions of these disaccharides with concanavalin A (con A) were characterized. The kinetics of binding of the galactopyranosyl-containing disaccharide to con A were found to be similar to those observed with monosaccharides in that monophasic time dependencies for binding were observed. The glucopyranosyl-containing disaccharide, however, exhibited biphasic time dependencies which were similar to those previously observed for the binding of p -nitrophenyl 2- O -α- d -mannopyranosyl-α- d -mannopyranoside to con A. These results support a model wherein the α-(1→2)-linked disaccharides which exhibit biphasic binding kinetics must be able to bind to con A in two different and mutually exclusive orientations. The ability to bind to con A in two orientations is shared by α-(1→2)-linked disaccharides in which both glycosyl residues can interact separately with the primary glycosyl binding site of con A. According to the model, the initial fast phase of the biphasic reaction reflects binding of the ligand in two orientations so that two complexes are formed in amounts determined by the relative values of the rate constants for formation of each complex. The subsequent slow phase is proposed to reflect a slow equilibration of the less stable complex to the thermodynamically more stable one. In the more stable complex, the glycosyl residue at the reducing end of the disaccharide occupies the primary glycosyl binding site. The added stability of this complex is attributed to extended interactions between con A and groups on the second glycosyl residue. An axial orientation of OH-2 of the second glycopyranosyl residue appears to be the most important determinant for the extended interaction.

The high level of proteolytic activity present in the midgut fluids of the detritus-feeding larvae of the crane fly, Tipula abdominalis, is due to a mixture of enzymes, consisting largely of serine proteases with trypsin- or chymotrypsin-like substrate specificities. These enzymes have high alkaline pH optima (pH > l l) and high stability at pH values of 8.5-11.5 at 15°C, properties that enable them to function effectively in the highly alkaline gut fluids (pH 9.5-11.5) of an insect that feeds and grows in cold water (0-25°C). The enzymes also have very low isoelectric points (pH < 6), resulting in their having a high negative charge in vivo. Possibly their anionic character prevents their inactivation through adsorption on ingested lignin, humic acid or clay. These serine enzymes arise from the midgut epithelial cells, and not from microbes residing in the gut or from ingested detrital food. The digestive system of T. abdominalis larvae is adapted for the efficient utilization of the limited quantities of dietary protein present in detrital food.

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.

High-performance liquid chromatography was used to analyze the kinetics of the thrombin-catalyzed release of the activation peptide from the factor XIII zymogen (fibrin-stabilizing factor). The specificity constant (kcat/Km) for this reaction, measured at factor XIII concentrations much below Km, was (0.13-0.16) X 10(6) M-1 s-1 at pH 7.4, mu = 0.15, and 37 degrees C. Separate estimates, obtained from the dependence of the initial rates of release of the activation peptide on the concentration of factor XIII, gave values of 10 (+/- 3) s-1 for kcat and 84 (+/- 30) microM for Km, in terms of ab protomers of the zymogen. The thrombin-mediated release of the activation peptide was dramatically enhanced in the presence of fibrinogen. Furthermore, the time course of release, in relation to that of fibrinopeptide A, suggested that some des-A-fibrinogen species (e.g., alpha 2B beta 2 gamma 2) may be the true activator for promoting the cleavage of the Arg-36 peptide bonds in the a subunits of factor XIII. This observation suggests that generation of factor XIIIa and its substrate (fibrin) is coordinated so that thrombin-mediated zymogen activation proceeds efficiently only after the process of clotting has been initiated by the removal of fibrinopeptide A from fibrinogen.

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.

M N-2-hydroxyethylpipetazine-N’-3-propesulfonic acid, pH 7.8, at 37°C for 15 mitt, and then determining the o-serine dehydratase activity of an aliquot of the incubation mixture. A lactic dehydrogenase-coupled assay (at 25°C) was used to measure the rate of enzymicahy catalyzed conversion of D-serine to pyruvate, wherein depletion of NADH was followed continuously at 340 nm. The concentration of pyridoxal Y-phosphate in the plasma samule was estimated from the enzymic activity which is a linear function of the amount of pyridoxal 5’-phosphate present in the assay.

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5′-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G—-D variants for pyridoxal 5′-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G—-D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5′-phosphate and for a pyridoxal 5′-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5′-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.

The equilibrium constant (KX) for the reaction D-serine dehydratase + pyridoxamine-P in equilibrium KX D-serine apodehydratase: pyridoxamine-P + pyridoxal-P was determined. At 25 degreees, pH 7.80, KX increases from 5.4 times 10-minus 5 to 21 times 10-minus 5 as T/2 is increased from 0.33 to 0.66. A value of 1.3 times 10-minus 4 M at 25 degrees, pH 7.80, T/2 0.33 for the equilibrium constant (KPMP) for dissociation of pyridoxamine-P from D-serine apodehydratase was determined from the ratio of the equilibrium constant for dissociation of pyridoxal-P from holoenzyme to KX. Pyridoxamine-P and the thiazolidine, formed from pyridoxal-P and cysteine, were found to have similar affinities for D-serine apodehydratase. Using the affinities of these derivatives as a measure of the noncovalent interactions between cofactor and protein, it was possible to estimate the contribution of the Schiff base linkage to the stability of the complex formed between pyridoxal-P and protein. The covalent Schiff base linkage in the holoenzyme was found to be no more stable than the Schiff base linkage formed between 6-aminocaproic acid and pyridoxal-P. The contribution of noncovalent interactions to the stability of the cofactor-protein complex was shown to be at least 20 to 40 times greater than the contribution of the covalent Schiff base linkage.

A new congenital dysfibrinogenemia, designated fibrinogen Petoskey, which was traced through four generations of a Michigan family, was found to exhibit an abnormally slow rate of release of fibrino-peptide A upon treatment with thrombin and batroxobin. Batroxobin only partially hydrolyzed and polymerized the fibrinogen from affected individuals, suggesting that these patients had both normal and abnormal fibrinogen in their circulation and that batroxobin was not capable of releasing fibrinopeptide A from the abnormal fibrinogen. Polymerization of fibrin monomers from fibrinogen Petoskey and plasmin mediated digestion of fibrinogen Petoskey were normal. The Factor XIIIa-catalyzed cross-linking of fibrinogen Petoskey was slightly delayed at low (but not at high) concentrations of thrombin. This delayed cross-linking appeared to be a secondary effect of the lower rate of release of fibrinopeptide A.

We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli. This was accomplished by isolating and sequencing the structural gene from wild type E. coli and from two mutant strains that produce inactive D-serine dehydratase. DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant. The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme. The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.

SummaryA kinetic analysis was developed to determine the steady state kinetic parameter kcat/KM for the thrombin-catalyzed release of FPA from abnormal and normal fibrinogen in mixtures of the two. Such mixtures are likely to comprise the fibrinogen of individuals with congenital dysfibrinogenemia. The analysis was used to characterize fibrinogen Grand Rapids, a new congenital dysfibrinogenemia. It indicated that fibrinogen from affected individuals was composed of normal and abnormal fibrinogen in roughly equal amounts, and that the value of kcat/KM for the thrombin-catalyzed release of FPA from the fibrinogen variant was 77fold lower than that for the release of FPA from the normal fibrinogen. In separate studies, fibrinogen Grand Rapids was found to exhibit a reduced clottability. Additionally, affected individuals appeared to have plasma fibrinogen concentrations which were about one-third the normal value.

Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

The carbohydrate binding sites of the jack bean lectin, concanavalin A, were studied spectrophotometrically. A number of ligands were examined for their ability to displace the chromogenic ligand p-nitrophenyl α-d-mannopyranoside. It is shown that this technique provides a simple and convenient approach for obtaining association constants for a series of displacing ligands. The data can best be interpreted in terms of a single carbohydrate binding site per monomeric unit for the protein in solution.

The base-catalyzed hydrolysis rate of the cationic esters obtained by quaternization of N,N,N′,N′-tetramethylethylenediamine with phenylbromoacetate was studied in buffers and in solutions of partially neutralized polymeric acids. The polyanions were found to be powerful inhibitors of the reaction. This effect was interpreted as due to the association of the ester with the polyanion, from which the catalytically active hydroxyl groups are repelled. It is shown that kinetic data may be interpreted by a procedure similar to the Lineweaver-Burk plot to yield the reactivity of the bound ester and the dissociation constant of the polymer–ester complex.

1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC) was shown to inactivate α-chymotrypsin. The close correspondence between efficiency and all or none assays during the inactivation reaction shows that CMC reacts with α-chymotrypsin to produce a derivative completely devoid of catalytic activity and that inactivation is probably caused by a reaction between CMC and a single aminoacyl residue on the enzyme. The dependence of the pseudo-first order rate constant for inactivation on the concentration of CMC and indole indicates that CMC interacts with the substrate-binding site of α-chymotrypsin and forms an adsorption complex with the enzyme before the inactivation reaction. At pH 7 and 25°, the first order rate constant (k2) for the formation of inactivated enzyme from the adsorption complex is 0.10 min-1, and the dissociation constant (Kd) for the adsorption complex is 0.013 m. The following results secured the conclusion that a seryl residue at the active site of α-chymotrypsin adds to CMC or 14C-CMC (1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-14C-p-toluenesulfonate) to form a catalytically inactive isoureylene derivative of the enzyme (CMC-chymotrypsin or 14C-CMC-chymotrypsin). (a) The inactivation reaction is associated with the incorporation into protein of 1 mole of 14C-CMC per mole of enzyme inactivated. (b) 2-Mercaptoethylamine displaces CMC (probably as 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)ureylene metho-p-toluenesulfonate from CMC-chymotrypsin to yield 1 S-aminoethylcysteinyl residue per inactivated enzyme molecule. (c) On standing in 5% acetic acid, 14C-CMC-chymotrypsin liberates radiochemical label, and the amino group of a seryl residue is exposed. (d) NH2OH reacts with 14C-CMC-chymotrypsin, regenerating active enzyme and liberating radiochemical label from protein in roughly equivalent amounts. (e) 14C-CMC is bound to an active center peptide obtained by digesting 14C-CMC-chymotrypsin with pepsin. In order to establish precedent for the reaction between 2-mercaptoethylamine and CMC-chymotrypsin, O-methylisourea was shown to S-methylate cysteine.

By studying the glutamate dehydrogenase catalyzed oxidation of glutamic acid under conditions where the concentration of enzyme exceeded that of NAD, it was possible to evaluate directly the first-order rate constant (0.73 sec−1) for the decay of an intermediate associated with the burst in absorbance, which is seen at 340 nm in the pre-steady state phase when the NAD concentration exceeded that of enzyme. A study of the wavelength dependence of the absorbance vs time plots when [enzyme] > [NAD] indicated that the intermediate associated with the burst is isosbestic with the ternary abortive complex E · Glu · NADH. This information made it possible to estimate the total amount of NADH (π) formed in the initial rapid phase when [NAD] > [enzyme]. A study of the dependence of the pre-steady-state kinetics on [NAD] revealed that between 27 and 1000 μM NAD, π was invariant at 0.4 μmol NADH formed in the burst phase per polypeptide chain. However, the velocity of the phase immediately following the burst phase increased between 27 and 1000 μM NAD, and the pseudo-first-order rate constant for the burst phase increased between 30 and 300 μM NAD and decreased between 300 and 1000 μM NAD. These results were taken to mean that all the NAD sites on glutamate dehydrogenase are not catalytically equivalent and that binding of NAD to sites which are not catalytically active causes a decrease in the rate of the burst phase and an increase in the rate of the phase following the burst. Below 1000 μM glutamate, a pronounced lag in the onset of the burst phase is seen. This lag suggests that at low glutamate concentration binding of NAD and/or glutamate to the enzyme becomes slower than reduction of E · Glu · NAD.

La complexation du polyacide marque avec le polyether conduit a une forte augmentation de l'intensite de fluorescence

The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kappacat/Km) for alpha-thrombin-catalyzed AP release, 1.2 x 10(5) M-1s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T.J., Lewis, S. D., Lorand, L., & Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder alpha-thrombin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP has no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure and physical properties of human fibrinogen and fibrin are reviewed along with methods for the detection of products of their metabolism. Interactions of human fibrinogen with thrombin, factor XIII, plasminogen, glycoprotein IIb/IIIa, and other proteins are related to their relevance to thrombosis and hemostasis. To the extent information is available, the structural determinants of these interactions are delineated, and kinetic and thermodynamic parameters associated with the interactions are listed. Individual steps in the reaction pathway for the conversion of fibrinogen to cross-linked fibrin are characterized. The altered hemostatic properties of mutational variants of fibrinogen are related to their altered structure. The structures of the genes coding for the polypeptide chains of fibrinogen are discussed along with the current state of knowledge of the control and regulation of fibrinogen synthesis. Fibrinogen catabolism and fibrinolysis are also reviewed.

The p K perturbations exhibited by nitrophenol reporter groups linked to the active site thiol group in papain were compared in order to probe the pH dependence of the charge in the environment of the active site of this enzyme. Nitrophenol reporter groups were introduced at the active site of papain by reaction of the active site thiol group with 4-chloromercuri-2-nitrophenol ( 1a ), 2-chloromercuri-4-nitrophenol ( 2a ), and 2-chloromercuri-4,6-dinitrophenol ( 3a ) to form papain derivatives 1c–3c . Spectrophotometric titrations of these derivatives of papain yielded p K values (25 °C, Γ 2 0.15) of 7.51, 7.99, and 3.93, for the reporter groups in 1c, 2c and 3c , respectively. Comparison of these p K values with those of the corresponding 2-mercaptoethanol derivatives 1b–3b indicates that the environment at the active site of papain caused upward perturbations of 0.35 and 0.96 unit in the p K 's of the reporter groups in 1c and 2c , respectively, and a downward perturbation of 0.53 unit in the p K of the reporter group in 3c . These p K perturbations were compared to those determined previously for the nitrophenol reporter groups introduced at the active site of papain by reaction of papain with α-bromo-4-hydroxy-3-nitroacetophenone and 2-bromoacetamido-4-nitrophenol. Assuming that the phenolic function of geometrically similar reporter groups occupy similar positions at the active site of the enzyme, the observed p K perturbations exhibited by these five reporter groups indicate that the charge in the vicinity of the active site of papain (a) does not change between pH 5 and 8, and (b) becomes positive at pH values below pH 4–5. These conclusions are consistent with the view that the p K values of His-159 and Asp-158 at the active site of papain are both about 4 in forms of papain wherein the active site thiolate anion is neutralized by protonation or is blocked by chemical modification.

The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.

The preparation of a reagent which can be used to attach biotin to proteins via a cleavable connector arm is described. The reagent, 3-(4-( N -biotinoyl-6-aminocaproyloxy)phenyl)propionic acid N -hydroxysuccinimide ester (BPE), can acylate amino groups of proteins and other compounds via reaction of the N -hydroxysuccinimide ester function. Compounds which have been covalently labeled with BPE can be selectively retrieved (at physiological pH values) from complex mixtures by adsorbing the target compounds on immobilized avidin. These compounds can be released from the avidin by cleavage of the phenyl ester linkage of the connector arm under conditions which don't denature proteins. Insulin was labeled with BPE. The binding of the labeled insulin to immobilized avidin, and its subsequent release by treatment at room temperature with 1 m hydroxylamine at pH 7 was demonstrated.

Papain was succinylated in order to increase its solubility above pH 8 so that proton NMR spectroscopy could be used to study the ionization of His-159 at the active site of the enzyme. The pH dependence of NMR spectra of catalytically active succinyl-papain and the methylthio derivative of the active-site cysteinyl residue of succinyl-papain (succinyl-papain-S-SCH3) were determined between pH 6 and 10. The pH dependence of the C epsilon 1 H resonance of His-159 in catalytically active succinyl-papain indicates that His-159 has a pK of about 8.6 in the catalytically active form of the enzyme. The position of this resonance in succinyl-papain-S-SCH3 indicates that when the active-site cysteinyl residue is methylthiolated, His-159 is completely deprotonated between pH 6 and 10. This result is taken as evidence for an imidazolium--thiolate ion-pair interaction between His-159 and Cys-25 wherein neutralization of the charge on the thiolate anion by methylthiolation would be expected to cause a marked decrease in the pK of His-159. A possible catalytic role for the ion pair in the acylation step in papain-catalyzed reactions is proposed wherein attack of a substrate by the imidazolium--thiolate ion pair is accompanied by an increase in the acidity of the imidazolium group that facilitates expulsion of the leaving group of the substrate.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-( N -succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N -succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.

Insulin receptor partially purified from human placenta by chromatography on immobilized wheat germ agglutinin was subjected to affinity cross linking to determine the relationship between the subunit structure of the multiple forms of the insulin receptor and their competence to bind insulin and undergo autophosphorylation. It was demonstrated that, whereas the 340-kDa intact receptor undergoes autophosphorylation, the 290- and 320-kDa insulin binding forms of the receptor do not. Phosphorylation at tyrosyl residues in the intact receptor was verified using a new facile method for determination of phosphorylated amino acids. The competence of the phosphorylated 340-kDa protein to bind insulin was demonstrated using a double-probe labeling protocol wherein receptor phosphorylated with [gamma-32P]ATP was cross-linked with disuccinimidyl suberate (DSS) in the presence of N epsilon B29-biotinylinsulin. The observation that succinylavidin, by virtue of its interaction with biotinyl residues, decreased the electrophoretic mobility of receptor radiochemically labeled with 32P indicated that the phosphorylated 340-kDa protein was competent to bind insulin. This result is compelling evidence that the 340-kDa phosphorylated species is insulin receptor itself, rather than a closely associated contaminant. Treatment of the receptor with the crosslinking agent DSS produced (after reduction and denaturation) alpha-dimer, beta-dimer, and a smaller amount of tetramer. This observation is consistent with a symmetrical, tetrameric, alpha 2 beta 2 structure for insulin receptor from human placenta, and excludes previously proposed alternative structures containing one alpha and one beta chain.

The recent availability of growth hormone (GH) produced by recombinant DNA techniques has greatly facilitated our ability to study the mechanism of action of GH. Nevertheless, we have made only limited progress in this regard, despite the fact that GH was first identified over 60 years ago. Progress has been difficult in part because GH has diverse actions, including growth promotion, induction of diabetes and stimulation of lipolysis (for recent reviews see Davidson 1987; Green et al. 1985; Isaksson et al. 1985). In addition, suitable cellular systems for analyzing GH action have been lacking. While rat adipose tissue and cells have been among the most sensitive in vitrotargets for GH, these preparations have limited viability in vitro(hours), while the physiological effects of GH are typically delayed in onset (days). Accordingly, several laboratories have used cell culture techniques to gain insight into delayed actions of growth hormone in adipocytes (Nyberg and Smith 1977; Maloff et al. 1980; Schwartz 1984; Walton et al. 1986; Doglio et al. 1986).