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Summary A glucagon analog with the following sequence has been synthesized: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Leu-Gln-Glu-Phe-Leu-Gln-Trp-Ala-Leu-Gln-Thr. When interacting with rat hepatocytes, the analog mimics, in part, the activities of glucagon in receptor binding and inhibition of carbohydrate incorporation into glycogen. Comparison of the binding of the analog with that of glucagon demonstrates the existence of two distinct homogeneous populations of glucagon receptors. The synthetic analog acts as a specific probe for those receptors that have a high affinity for glucagon.

Lipoprotein lipase from bovine milk reacted stoichiometrically with diisopropylphosphorofluoridate (DFP), an inactivator of serine esterases, resulting in the loss of enzymatic activity against triacylglycerols. The reaction obeyed first-order kinetics with a rate constant of 0.69 h-1. In order to isolate the peptide containing the diisopropylphosphoryl moiety (DIP), partially purified lipoprotein lipase was covalently labeled with [3H]DFP, and the labeled protein was reduced, carboxymethylated, and further purified to about 90% homogeneity. Cyanogen bromide cleavage followed by gel filtration yielded a radioactive peptide of 6-8 kDa. This peptide was succinylated and then digested with Staphylococcus aureus V8 proteinase. From this digest, a peptide containing 0.95 mol of [3H] DIP/mol of peptide was isolated by gel-permeation chromatography followed by reverse-phase high performance liquid chromatography. Automated Edman degradation provided the following sequence: Ala-Ile-Gly-Ile-His-Trp-Gly-Gly- (DIP)Ser-Pro-Asn-Gln-Lys-Asn-Gly-Ala-Val-Phe-Ile-Asn-(Ser, Leu)-Glu. Analysis of the sequence for secondary structure suggests that the reactive serine of lipoprotein lipase is in a beta-turn, a structure similar to those of the active sites of most other serine proteinases. Lipoprotein lipase appears to share this secondary structure with other serine hydrolases despite significant differences in the primary structure of this domain.

The enzymatic reaction of porcine pancreatic lipase with insoluble monolayers of either trioctanoin or 1,2-dioctanoin can be followed quantitatively by monitoring the considerable change in surface pressure which occurs during the course of the reaction. The rate of each reaction is proportional to the surface concentration of the substrate. The calculated pseudo-first order rate constants are proportional to the enzyme concentration in the bulk solution. At pH 7.6, the value of the second order rate constant for the hydrolysis of the primary ester function in trioctanoin was found to be 8.46 x 105 m-1 s-1, and that for dioctanoin, 2.65 x 104 m-1 s-1. The pH dependency of the rate constant for dioctanoin hydrolysis reveals an ionizable basic group at the active site of the enzyme with pKa of 6.38. The results are consistent with fully hydrated enzyme molecules acting upon substrate molecules lying within the insoluble mono-layer.

Porcine pancreatic phospholipase A2 was purified from commercial pancreatin by a method involving heat denaturation, trichloroacetic acid precipitation, and DEAE-cellulose chromatography. Assaying the eluate of the chromatography step by a new titrimetric method using vegetable lecithin-albumin emulsion as the substrate, several species of phospholipase A were found. Some of these went undetected when the conventional egg yolk emulsion assay was used. Two phospholipases A2 were isolated in a homogeneous form and shown to have similar chemical and physical properties. Catalytic specificity of the two enzymes differs remarkably toward lecithins in different emulsified states.

The rate constant of the glyoxylate carboligase-catalyzed condensation of glyoxylate has been measured at high enzyme concentrations. A saturation effect has been observed: the rate constant approaches a limiting value independent of enzyme concentration. Kinetic analysis shows that at these high enzymic rates, the rate-limiting step becomes the nonenzymic dehydration of the extensively hydrated aldehyde function of glyoxylate. The value of the rate constant measured in this way ( k dehyd = 5.0 × 10 −2 sec −1 at 25 °, pH 7.8) is in agreement with previously published estimates for the dehydration of hydrated glyoxylate. Thus, the enzyme has a high specificity for unhydrated glyoxylate, and does not catalyze the dehydration reaction.

The pH dependence of the α-chymotrypsin-catalyzed hydrolysis of l-tryptophan methyl ester in H2O and D2O and of d-tryptophan methyl ester was determined by spectroscopic means (kcat, Km and kcat/Km). No product inhibition or substrate activation was observed. The kinetic parameters of the α-chymotrypsin-catalyzed hydrolysis of l-tyrosine p-nitrophenyl ester were also determined. Values of kcat for both the tyrosine and tryptophan derivatives depend on the ionization of the amino group. For the tryptophan derivatives which were investigated in most detail, the pH dependence of kcat/Km ( = k2/Ks) yields a bell-shaped curve with a maximum at pH 7.2. The deuterium oxide kinetic solvent isotope effect and the dependency of the reaction on a base of pK ∼ 7 for the l derivative are comparable to that seen with the corresponding acylamino derivative. Thus, the mechanism of hydrolysis of the amino acid esters appear to be the same as that for the acylamino acid esters. However, the ionizability of the α-amino group in the former produces a markedly different pH dependency of the enzyme-catalyzed reaction, which can be attributed to the presence of a protonated and unprotonated substrate. The protonated substrate is about 20-fold faster than the unprotonated substrate in both acylation and deacylation of l-tryptophan methyl ester. Full specificity of the enzyme (l over d) is found for l-tryptophan methyl ester in its protonated form. This may be due to a hydrogen bond between the substrate and the enzyme with very strict orientational requirements.

The reaction of porcine pancreatic lipase B (EC 3.1.1.3) with the soluble triglyceride, tripropionin, shows substantial stimulation in the presence of hydrophobic surfaces. The enhancement of the enzymatic rate can be correlated with the reversible binding of the enzyme to the hydrophobic surface with a dissociation constant K = 1.3 x 10-8 m. The binding of the enzyme to the surface is diffusion controlled with a rate constant of 1.8 x 106 m-1 s-1. At saturation of the surface with enzyme each protein molecule occupies an average area of 6000 A2 per molecule. The hydrolytic reaction on the surface is first order with respect to the amount of adsorbed enzyme and the concentration of tripropionin at the solution-solid interface. The second order rate constant of the reaction is 1.3 x 1013 cm2 mole-1 s-1. The surface reaction requires a basic group on the enzyme with a pK of 5.85. The enhancement of the velocity on the surface as compared to the homogeneous reaction can be ascribed to an increased local concentration of the substrate at the liquid-solid interface.

An acidimetric method is proposed to distinguish between carbon dioxide and carbonic acid as the primary product of enzymic decarboxylation reactions. Application of this method to the thiamine pyrophosphate-requiring Escherichia coli glyoxylate carboligase reaction (the decarboxylating condensation of glyoxylate to yield hydroxymalonic semialdehyde) at pH 8 shows a transient disappearance of protons. Kinetic analysis of this proton uptake shows that the reaction occurs according to the pathway [for equation see PDF] The value of kh measured during the enzyme-catalyzed reaction is identical to the independently determined rate constant of carbon dioxide hydration. Thus, the intermediate is carbon dioxide, and the enzyme does not catalyze its hydration. The identity of the intermediate is further confirmed by the fact that addition of carbonic anhydrase to the reaction mixture suppresses the pH changes.

1. 1. The larval high density lipoprotein (HDL) from the hemolymph of Manduca sexta, isolated by density gradient centrifugation, contains 61% protein, 37% lipid and 2% carbohydrate. 2. 2. The molecular weight of HDL is 6 × 10 5 , with two apoproteins of 2.85 × 10 5 and 8.1 × 10 4 daltons. 3. 3. The large apoprotein is destroyed by trypsin treatment of the particle, while the small one is not. 4. 4. Calculations based upon size and composition show that this particle is very different in structure from mammalian lipoproteins. It is proposed that a portion of the apoprotein occupies the central core region.

Monoclonal antibodies against human apolipoprotein A-I (apoA-I) were generated by the hybridoma technique. Clone G-10 was selected on the basis of its highest titer. The affinity of this antibody toward a series of synthetic peptides differing in length, amino acid composition, and amphiphilicity was tested by using both the indirect and the competitive enzyme-linked immunosorbent techniques (ELISA). From these measurements we calculated dissociation constants of the complexes of the antibody with apoA-I bound to the surface of the microtiter plate, apoA-I in solution, and any of the several peptides in solution. The dissociation constant (Kd) of the immobilized apoA-I/anti-apoA-I-complex, Kd = 2 x 10(-9) M, was significantly lower than that of the complex resulting from the interaction between anti-apoA-I and either apoA-I in solution or any of the several amphiphilic helical peptides in solution. Peptides devoid of amphiphilic secondary structure were inert. These data are consistent with the proposal that monoclonal G-10 recognizes in antigenic peptides an alpha-helical secondary structure of defined hydrophilic-lipophilic balance and comparatively less the specific amino acid side chains. We propose that the highest contribution to the free energy of binding (8 Kcal/mole) is derived from the docking of the helix to the antibody. It follows that in probing the specificity of a monoclonal antibody the conformation and the physical environment of the interacting antigen must be taken into account.

We have demonstrated that the lon+ (capR+) ATP-hydrolysis-dependent protease is inhibited by the addition of an enzymatically inactive mutant form of the protein (capR9 protein). The enzymatic activity of the capR+-capR9 protein mixture is also more stable to heat than the capR+ protein alone indicating that hybrid molecules are formed. Independent measurements demonstrated that the lon+ ATP-hydrolysis-dependent protease is a tetramer of identical 94 × 103 molecular weight monomers. The pattern of defective subunit (capR9) inhibition indicates only tetramers containing three capR9 monomers and one capR+ monomer are inactive, i.e. two or more capR+ monomers are required for a tetramer to act as an ATP-hydrolysis-dependent protease. Two molecular mechanisms to explain the dominance of a mutant allele, termed anticomplementation, are considered.

The 30S initiation complex, formed with the 30S ribosomal subunit, mRNA, and fMet-tRNA, has been shown by kinetic analysis with limiting concentrations of Escherichia coli ribosomes to be an obligatory intermediate in the formation of the 70S initiation complex. The formation of the 70S initiation complex began with an induction period and was proportional to the concentration of the 30S complex, which rapidly rose to a peak. The entire time course of the sequential pseudo-first-order, second-order reaction was reproduced accurately by the overall rate expression, in which we used rate constants that were determined by carrying out 30S and 70S complex formation separately. By using limiting concentrations of mRNA, we showed that phage MS2 RNA contained no specific signal that enhanced its rate of 30S complex formation with E. coli ribosomes and initiation factors; the pseudo-first-order rate constants obtained with poly(A3C9G1U1), poly(C15G1U4), and poly(G1U3) were 12-45 times higher than that with MS2 RNA. The observation that the rate constants for binding of fMet-tRNA and AcPhe-tRNA with a given synthetic RNA were comparable indicated that the initiator codon is recognized only indirectly through the initiator tRNA.

The activity of 3-hydroxy-3-methylglutaryl CoA reductase in homogenates of the corpora allata of the tobacco hornworm, Manduca sexta , was competitively inhibited by compactin. The KI for the sodium salt form of compactin was 0.9 nM for the reductase from both male and female corpora allata. In intact female corpora allata juvenile hormone biosynthesis was also inhibited by approximately 50 percent at 10 nM compactin. Following injection with compactin, darkening of the cuticle, an indication of juvenile hormone deficiency, was observed in larvae after ecdysis from third to fourth instar. Hence, compactin shows potential as an inhibitor of insect growth and development.

The two major acidic sulfhydryl proteases, bromelain A and bromelain B, from the pineapple stem have been characterized by their kinetic properties toward p-nitrophenyl esters of N-α-acyl amino acids. Both enzymes show the same broad specificity toward the amino acid side chains at pH 4.7. Less than an order of magnitude of difference is observed in the rates of bromelain A-catalyzed hydrolyses of the following five amino acid esters: Z-l-lysine (kcat = 7.4 s−1, Km = 57 μM), Z-l-alanine (kcat = 2.5 s−1, Km = 24 μm), Z-l-tyrosine (kcat = 0.4 s−1, Km = 7.6 μm), Z-glycine (kcat = 1.75 s−1, Km = 174μm) and Z-l-asparagine (kcat = 1.5 s−1, Km = 75 μm). Valine, leucine, isoleucine, and tryptophan derivatives react at least one-thousand times slower. The pineapple stem acetone powder also contains sulfhydryl proteases of markedly different specificity. A purification procedure of bromelain A and B is described which eliminates these enzymatic impurities.

The effect of tris (hydroxymethyl) aminomethane concentrations on the rate of calf intestinal alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate was studied, in the pH range 8-10, where no transphosphorylation reaction could be detected. Kinetic analysis of the results permitted description of the effect of Tris concentrations T on the rate of enzyme catalyzed hydrolysis (V) by the following equation: (see article). The rate-accelerating effect of Tris concentrations can be ascribed to two different mechanisms: At moderate Tris concentrations (0.01-0.20 M) the enzyme forms a reversible addition complex with a Tris molecule. This complex has an enhanced catalytic activity. We suggest that the binding of Tris to the enzyme could potentiate a second active site of the enzyme, due to its ionization effect upon an acidic group of the enzyme of pK = 8.9. The modest linear rate accelerating effect of Tris at high concentrations (0.20-0.60 M) could be ascribed to the change of the dielectric constant of the medium, the degree of solvation of the protein, or change in the tertiary structure of the enzyme.

Enzymatic catalysis has fascinated biochemists and physical and organic chemists alike for several generations. By their specificity and their catalytic efficiency enzymes are even today the paragons of homogeneous catalysis, especially when catalysis in aqueous media at neutral pH values is concerned. Thus, the mechanism of enzymatic catalysis is the subject of more intense study than ever before.

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.

Before discussing the structure of lipoproteins, a brief survey of the surface properties of their major constituents is in order. This will be followed by a discussion of the basic thermodynamic laws governing small spherical particles. The main discussion will be concerned with the gross structural features of lipoproteins, which can be predicted from the regularities observed in chemical composition as a function of particle size.

Spectrophotometric methods are used to explore the kinetics of ester hydrolysis by alpha-chymotrypsin.

Seven closely related protease inhibitors were isolated from commercial bromelain acetone powder in electrophoretically pure form by gel filtration on Sephadex G-75, followed by ion exchange chromatography on DEAE Sephadex at pH 7.55. The inhibitors are proteins of MW 5000-6000, which inhibit competitively the bromelaincatalyzed hydrolysis of CLN (Ki ≈ 10−7 M). This inhibition is optimal at pH 3 to 4,. and it depends upon the ionization of two acidic residues of pK = 4.5 and 5.0. In the acidic pH range the inhibitors are also effective toward papain, ficin and trypsin.