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Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW)(3) repeat sequence at the C terminus that consists of three tandem repeats (alpha, beta, and gamma). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp(444) in the alpha repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp(444) and the corresponding aspartate residues in the beta and the gamma repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1 RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the RNase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the templatetprimer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG) 10 complexes than for the poly (rA):(dT) 10 complexes.

The cholesteryl ester transfer protein-catalyzed cholesteryl ester transfer is inhibited by two compounds identified by a large-scale screening of cholesterol backbone-containing molecules. Kinetic analysis shows that U-95,594, an amino steroid, inhibits competitively the cholesteryl ester transfer protein-catalyzed transfer of both cholesteryl esters and triglycerides, as well from high-density lipoproteins as from synthetic microemulsions. In contrast, U-617, an organomercurial derivative of cholesterol, inhibits competitively the transfer of cholesteryl ester from either donor but is without any effect on triglyceride transfer. In addition to the rapid, competitive inhibition of cholesteryl ester transfer, U-617 also slowly and reversibly reacts with cholesteryl ester transfer protein to produce an additional 10-fold decrease in cholesteryl ester transfer activity but, again, without effect on triglyceride transfer.

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --exchange --desorption --diffusion.

cis - Parinaric acid (PnA), cis - trans - trans - cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (ϵ = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radiacals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78,517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10–15 times more potent than the aminosteroids evaluated (see Table 1).

The bisheteroarylpiperazine U-90152E is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound inhibits both the RNA- and DNA-directed DNA polymerase functions of HIV-1 RT. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-90152E. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived, which allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The results obtained indicate that U-90152E acts exclusively as a mixed inhibitor with respect to the template: primer and dNTP binding sites for both the RNA- and DNA-directed DNA polymerase domains of the enzyme. The inhibitor shows a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently does not directly impair the functions of the substrate binding sites. Therefore, U-90152E appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond.

Apo A-I, the major protein component of high density lipoprotein (HDL), is synthesized by hepatic and intestinal cells and assembled with lipids to produce, in as yet incompletely understood ways, a mature HDL particle. For many secreted proteins only a portion of newly synthesized polypeptides are secreted, with the remainder being degraded at intracellular sites. For example apolipoprotein B secretion is controlled by the extent of intracellular degradation of the protein. Here we have systematically examined whether there is significant intracellular degradation of nascent apo A-I. We find that in two hepatic cell types, primary cultures of hepatocytes from cynomolgus monkey and HepG2 hepatocarcinoma cells, essentially all apo A-I that is synthesized is eventually secreted. A non-hepatic cell line, Chinese hamster ovary cells transfected with the apo A-I gene, secreted somewhat less (65%) of the apo A-1 synthesized. In a careful kinetic analysis, the rate of apo A-I secretion was found to be identical between the three cell types. This indicates that the mechanisms governing secretion are conserved among the different cell types. Further, the rate of secretion was the same for apo A-I in a lipid-poor form and in a form found associated in the medium with sufficient lipid to promote flotation in density gradients. The kinetic analysis indicates that there are two rate limiting steps to apo A-I secretion from the cell. It has previously been suggested that, for most proteins, exit from the endoplasmic reticulum is the rate limiting step in the secretory pathway. However, the first of the measured rate constants does not correspond to previously measured rates of exit from the endoplasmic reticulum, indicating that exit from the endoplasmic reticulum is probably not rate limiting in apo A-I secretion. Together, these data suggest that in hepatic cells, contrary to what is observed for apo B secretion, intracellular degradation and transport are not limiting factors in apo A-I secretion.

Publisher Summary This chapter focuses on the chromogenic substrates and assay of phospholipase A 2 . The high specificity of phospholipases A 2 (PLA 2 ) toward aggregated substrates renders the assay of phospholipase A 2 a particularly challenging analytical task. Sensitive assays require the use of aggregated substrates, such as micelles, mixed micelles, single bilayer vesicles, or monomolecular layers, where the activity of the enzyme depends critically on the physical state and the exact composition of the nonaqueous phase. For reproducible assays with such heterogeneous systems, the experimental conditions must be strictly controlled as the presence of minor lipid impurities, the accumulation of small amounts of reaction products in the early phases of the reaction, or even slight changes in temperature or buffer composition may and do elicit large changes in the rate of the enzymatic reaction. Homogeneous reaction kinetics, which are conducive to readily reproducible kinetics, could only be achieved with short-chain lecithins, such as dibutyryllecithin, toward which phospholipases A 2 display a rather low specificity, and the assays based on these substrates are not sensitive enough for most purposes. The chapter describes a spectrophotometric assay, which lies between the two extremes, and which by virtue of its simplicity, is readily adaptable to a variety of purposes ranging from routine analyses to investigations of mechanistic details of the enzymatic reaction. This assay provides a rapid, accurate, and convenient method for measuring phospholipase A 2 concentrations as low as 1 × 10 -8 M.

To determine which functional groups of bases within the grooves of double-helical DNA interact with the HpaI endonuclease, we have employed chemically synthesized octanucleotides containing base analogues. The 5-methyl group of thymine was probed as a contact between the HpaI endonuclease and its recognition sequence by using the oligonucleotides d(G-G-T-T-A-A-C-C), d(G-G-T-U-A-A-C-C), and d(G-G-T-U(Br)-A-A-C-C). The 2-amino group of guanine was probed as a contact for the HpaI endonuclease by using the octanucleotide d(G-I-T-T-A-A-C-C). The HpaI endonuclease cleaves octanucleotides d(G-G-T-T-A-A-C-C) and d(G-G-T-B-A-A-C-C) according to Michaelis-Menten kinetics. However, both the Km and turnover number for d(G-G-T-B-A-A-C-C) were severalfold lower than those for cleavage of d(G-G-T-T-A-A-C-C). In addition, d(G-G-T-U-A-A-C-C) was not cleaved by HpaI endonuclease, suggesting that the 5-methyl group of thymine is a contact between the HpaI endonuclease and its recognition sequence. d(G-I-T-T-A-A-C-C) was not cleaved by the HpaI endonuclease which may be due in part to the low thermal stability of the duplex. Nevertheless, our results suggest that the 2-amino group of guanine is a contact for the HpaI endonuclease. A phosphate group 5' external to the HpaI recognition sequence has been identified as a contact between the HpaI endonuclease and DNA. The HpaI endonuclease cleaved 5'-phosphorylated octanucleotide 30-fold faster than unphosphorylated octanucleotide. In addition, the Km of the d(G-G-T-T-A-A-C-C) was 8000-fold higher than the Km of the phage f1 RFI DNA, suggesting that the octanucleotide is too short to take advantage of the entire DNA binding site of the enzyme.

In earlier studies, we prepared a docosapeptide, 1, designed with minimum homology as an amphiphilic alpha-helical model of apolipoprotein A-I (apo A-I) and described its lipid-binding characteristics, surface properties, and enzyme-activating ability. Although the affinity of 1 for egg lecithin unilamellar vesicles was comparable with that for the binding of apo A-I, the affinity of 1 for mixed lecithin/cholesterol (4:1 mol/mol) vesicles was less than that of apo A-I. It appeared possible that the 3-hydroxyl group of cholesterol may have a deleterious interaction with the hydrophobic portion of the amphiphilic helix of 1 that is inserted into the vesicles. Examination of the amphiphilic alpha-helical segments of apo A-I suggested that the preferential interaction of apo A-I with the mixed vesicles might be due to the presence of polar arginine residues in the otherwise hydrophobic regions of two of the helices. Therefore, we synthesized a model docosapeptide, 2, corresponding to the sequence of 1 but containing arginine rather than leucine at position 10 in the hydrophobic region of the alpha helix to assess the role of the alcohol function of cholesterol in protein--cholesterol interactions. The results of studies on the binding of 2 to unilamellar vesicles containing lecithin only, lecithin/cholesterol, lecithin/cholesterol hemisuccinate, or lecithin/cholesterol methyl ether were consistent with the postulate that the major role of cholesterol in the binding of proteins to phospholipid surfaces is the creation of free space between the phospholipid head groups that can accommodate the amphiphilic peptide chains at the interface.

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.

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.

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.

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.

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.

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.