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D Ullmann

Publications and source records attributed to D Ullmann.

17 recordsLinked to original sources
PubMed2000

Two-dimensional fluorescence intensity distribution analysis: theory and applications.

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.

Algorithms↗
PubMed1999

Fluorescence-intensity distribution analysis and its application in biomolecular detection technology.

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5'-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.

DNA↗
PubMed1999

S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.

Binding Sites↗
PubMed1998

Design and synthesis of fluorogenic trypsin peptide substrates based on resonance energy transfer.

An assay based on new internally quenched fluorogenic peptide substrates with the general structure 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)-Gly-Pro-Ala-Xaa-Leu-Ala-Ile-Gly-5-(2-aminoethylamino++ +)naphtha lene-1-sulfonic acid (EDANS), where Xaa = Arg, Lys, has been developed to measure proteolytic activity of trypsin and similar proteases. The kinetic parameters for the tryptic hydrolysis of DABCYL-Gly-Pro-Ala-Arg-Leu-Ala-Ile-Gly-EDANS are Km = 34 microM, kcat = 40 s-1, and kcat/Km = 1.17 x 10(6) M-1 s-1. The substrates offer two advantages over common substrates. First they are very sensitive. Applications to chemically modified trypsin and engineered variants show the ability to detect traces of proteolytic activity. In addition, these substrates are adapted to the S'-specificity of the investigated protease. These features and the prospect of miniaturization makes the assay suitable for applications to high-throughput screening.

Amino Acid Sequence↗
PubMed1998

Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues.

The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with Glu and Asp to introduce a high specificity for basic residues into the S1' site of trypsin. Both mutations cause a dramatic increase in the S1' specificity for Arg and Lys as measured by acyl transfer reactions. In K60E, the preference for Arg increases 70-fold while the preference for P1'-Lys increases 12-fold. In contrast, the preferences for other P1' residues either decrease slightly or remain the same. Thus, K60E prefers P1'-Arg over most other P1' residues by 2 orders of magnitude. Similar results are obtained when P1' specificity is measured in peptide cleavage assays. K60D exhibits an S1' specificity profile very similar to that of K60E, although the P1'-Arg preference is reduced by a factor of 2.5. Molecular modeling studies suggest that the high S1' specificity for Arg in K60E may be due to the formation of a salt bridge between Glu60 and the P1'-Arg of the substrate.

Amino Acids, Diamino↗
PubMed1997

Converting trypsin to chymotrypsin: structural determinants of S1' specificity.

Trypsin and chymotrypsin differ strikingly in substrate specificities despite great similarity in their primary and tertiary structures. This work analyzes the role of two surface loops, loop 40 and loop 60, as structural determinants of the specificity of the S1'-subsite in chymotrypsin and trypsin. Chymotrypsin prefers P1' Arg/Lys residues, while trypsin prefers hydrophobic P1' residues. We replaced loop 40 and loop 60 in trypsin with their chymotrypsin counterparts. These mutations do not affect the S1 specificity and catalytic activity of trypsin. The S1' specificity was analyzed by monitoring acyl-transfer reactions to 16 amino acid amides. The exchange of loop 40 does not affect the S1' specificity. In contrast, the replacement of loop 60 causes a loss of specificity for P1'-Met/Ile/Leu. Combining both mutations reconstitutes a chymotrypsin-like S1' specificity. The specificity for Arg-NH2 increases 3-fold while the preferences for Met-NH2 and Ile-NH2 decrease 4- and 8-fold, respectively. Therefore, P1'-Arg/Met discrimination changes by factor 12 and P1'-Arg/Ile discrimination changes by factor 24. Thus, loop 40 and loop 60 act synergistically to determine S1' specificity in trypsin and chymotrypsin.

Amino Acid Sequence↗
PubMed1997

Subsite specificity studies on the unusual cysteine protease clostripain: charged residues in the P3 position indicate a narrow subsite region.

The importance of electrostatic interactions between charged residues at the P3 position of substrates and the S3 subsite of the cysteine protease clostripain was investigated. For this purpose quantitative enzymatic hydrolysis studies using steady state kinetics have been carried out within a set of N alpha-protected synthetic dipeptide ester substrates with systematic changes of their charge in the P3 position. It was demonstrated that, in contrast to the former postulated second anionic S3 subsite, the lowest specificity was for the hydrolysis of the positively charged substrates. However, this effect was strongly dependent on the individual amino acid at P1. Furthermore, we investigated how far these P3-S3 interactions reflect on the S' subsite specificity via acyl transfers. Apart from the general weak influence of the charge at P3 on the deacylation kinetics, nucleophiles with proline at P'1 play an extraordinary role. Surprisingly, in contrast to the poor primary lysine specificity, acyl transfer using P1 lysine substrates does not affect the nucleophile efficiency found with the corresponding arginine substrates.

Acylation↗
PubMed1994

The specificity of clostripain from Clostridium histolyticum. Mapping the S' subsites via acyl transfer to amino acid amides and peptides.

The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using N alpha-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-Xaa-Ala-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P'1 position, low specificity for amino acid amides and Xaa-(Ala)3-Gly peptides was found, however, in the P'2 position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P'1 and P'2 positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P'1-P'3 increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of P'3Phe. Surprisingly, P'1Gly leads to effective P'-S' interactions. However, the opposite result was obtained for P'2Gly. The high efficiency for Gly-NH2 does not fit with the hydrophobicity structure/activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH2 with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P'1Pro and P'2Gly and a negative cooperativity between P'1Pro and P'2Phe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.

Acylation↗
PubMed1994

S'-subsite mapping of polyethylene glycol-modified alpha-chymotrypsin and alpha-chymotrypsin: a comparative study.

Nucleophile specificities of polyethylene glycol-modified alpha-chymotrypsin and the native enzyme were investigated via acyl transfer reactions using Ac-Tyr-OEt as acyl donor and a large series of peptides and amino-acid amides as nucleophiles. In acyl transfer reactions with amino-acid amides both enzymes prefer basic and bulky amino-acid residues. However, peptides with bulky aliphatic or aromatic residues in P1' position were very poor nucleophiles for both enzymes. Surprisingly, peptides having bulky aliphatic or aromatic residues in P2' were preferred by the modified enzyme and were apparently more efficient nucleophiles for both enzymes than those with such residues in P1'. Generally, peptides with a longer chain were weaker nucleophiles in the reactions catalyzed by polyethylene glycol-modified enzyme. In the series of peptides containing a positively charged amino-acid residue in various locations, the order of nucleophilic efficiency is with this location being: P1' > P3' > P2'; this is valid for both enzymes.

Amino Acid Sequence↗
PubMed1994

Kinetic characterization of affinity chromatography purified clostripain.

The cysteine protease clostripain, purified by affinity chromatography on a large scale, shows very high activity against BAEE using the titrimetric standard assay. Furthermore, titration of the active site with the irreversible inhibitor tosyl-lysyl-chloromethane resulted in a more than two times higher specific activity compared with literature data (Porter et al. (1971) J. Biol. Chem. 246, 7675-7682). Based on the molar enzyme concentration determined, the hydrolysis kinetics of the standard substrate BAEE were compared with those for the N alpha-protected dipeptide ester Mal-Tyr-Arg-OEt. It was demonstrated from the kinetic data that the highly purified clostripain is the most active enzyme preparation available up to now. In contrast to the standard substrate, Mal-Tyr-Arg-OEt shows a threefold lower specificity constant.

Arginine↗
PubMed1993

The hypotriglyceridemic effect of fish oil in adult-onset diabetes without adverse glucose control.

Diabetic control as judged by five criteria did not deteriorate after 6 months of fish oil compared to 6 months of olive oil supplementation in 16 patients with NIDDM who were eating a low fat, high complex carbohydrate diet. Plasma total and VLDL triglyceride and cholesterol decreased significantly after fish oil supplementation; plasma total and HDL cholesterol concentrations did not change. The LDL cholesterol level was significantly increased with fish oil supplementation, suggesting that patients with NIDDM who are given a fish oil supplement to decrease the plasma total and VLDL triglyceride levels may also need further dietary and/or pharmaceutical therapy to maintain an LDL cholesterol level compatible with a low risk of coronary disease. The study emphasizes the safe use of fish oil over a 6-month period in diabetic patients.

Aged↗
PubMed1992

A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians.

OBJECTIVE: To assess prospectively the risk of coronary heart disease associated with elevated plasma levels of homocyst(e)ine. DESIGN: Nested case-control study using prospectively collected blood samples. SETTING: Participants in the Physicians' Health Study. PARTICIPANTS: A total of 14,916 male physicians, aged 40 to 84 years, with no prior myocardial infarction (MI) or stroke provided plasma samples at baseline and were followed up for 5 years. Samples from 271 men who subsequently developed MI were analyzed for homocyst(e)ine levels together with paired controls, matched by age and smoking. MAIN OUTCOME MEASURE: Acute MI or death due to coronary disease. RESULTS: Levels of homocyst(e)ine were higher in cases than in controls (11.1 +/- 4.0 [SD] vs 10.5 +/- 2.8 nmol/mL; P = .03). The difference was attributable to an excess of high values among men who later had MIs. The relative risk for the highest 5% vs the bottom 90% of homocyst(e)ine levels was 3.1 (95% confidence interval, 1.4 to 6.9; P = .005). After additional adjustment for diabetes, hypertension, aspirin assignment, Quetelet's Index, and total/high-density lipoprotein cholesterol, this relative risk was 3.4 (95% confidence interval, 1.3 to 8.8) (P = .01). Thirteen controls and 31 cases (11%) had values above the 95th percentile of the controls. CONCLUSIONS: Moderately high levels of plasma homocyst(e)ine are associated with subsequent risk of MI independent of other coronary risk factors. Because high levels can often be easily treated with vitamin supplements, homocyst(e)ine may be an independent, modifiable risk factor.

Adult↗
PubMed1991

Cause-specific mortality among physicians with differing life-styles.

We compare the mortality experience of medical school graduates from Loma Linda University (LLU [n = 4342]) and the University of Southern California (USC [n = 2832]) with each other and with that of contemporaneous, white, American men. When compared with US white men from the general population, both USC and LLU graduates had below expected deaths for all causes (USC, standardized mortality ratio [SMR] = 76; LLU, SMR = 56), although deaths due to cerebrovascular disease, airplane accidents, and suicides were elevated for USC (SMRs = 132, 360, and 218, respectively). The LLU graduates had a risk similar to that of the USC graduates for fatal cancer, with a mortality ratio (MR) of 0.92 (95% confidence interval, 0.67 to 1.26); but half the risk of fatal atherosclerotic disease, with MRs of 0.58 (0.46 to 0.73) and 0.66 (0.43 to 0.99) for coronary and cerebrovascular disease, respectively; and three times the risk of fatal airplane accidents. The overall mortality rate of LLU physicians was only 75% as high as that of the USC physicians and only 56% as high as that of the US male population at large. We attribute this reduced mortality mainly to the low cardiovascular mortality rates, which may be accounted for by the life-style of the substantial proportion of Seventh-day Adventists among LLU graduates.

Accidents↗
PubMedDate not supplied

Will a high-carbohydrate, low-fat diet lower plasma lipids and lipoproteins without producing hypertriglyceridemia?

A sudden increase in dietary carbohydrate invariably increases the plasma levels of very low density lipoprotein (VLDL) and triglyceride. The present studies were designed to test the hypothesis that dietary carbohydrate-induced hypertriglyceridemia need not occur. In the first study we fed gradually increasing amounts of carbohydrate and gradually decreasing amounts of fat to eight subjects. The usual American diet (40% fat, 45% carbohydrate, and 15% protein) was followed in sequence by four diets in a phased regimen, the carbohydrate increasing by 5% of total calories and the fat content decreasing by 5% for each dietary period. In the last dietary period (phase 4), 20% of the energy was in the form of fat and 65% in the form of carbohydrates; the cholesterol content was 100 mg/day. Throughout the study, plasma triglyceride and VLDL triglyceride levels did not change significantly. The plasma total and low density lipoprotein (LDL) cholesterol levels were greatly reduced, by 15% and 22%, respectively (p = 0.004). Plasma high density lipoprotein (HDL) cholesterol levels decreased concomitantly. In the second study, after a washout period six of the subjects were initially fed the phase 4 high-carbohydrate diet for a 10-day period. The plasma triglyceride concentration increased over baseline levels by 47%, and VLDL triglyceride levels increased by 73%. We conclude that although a sudden increase in dietary carbohydrate increases the plasma triglyceride level, patients gradually introduced to a high-carbohydrate, low-fat diet may achieve a significant reduction of plasma total and LDL cholesterol without developing carbohydrate-induced hypertriglyceridemia.

Adult↗
PubMedDate not supplied

Non-conventional enzyme catalysis: application of proteases and zymogens in biotransformations.

One of the attractions of using enzymes for chemical syntheses is the control of stereochemistry: problems of racemization that attend chemical C-N ligation methods are completely avoided. Furthermore, the enzymatic approach has the advantage that only minimal protection-deprotection steps are involved. The Impetus to develop non-conventional catalysis procedures has sprung from the lack of usable native enzymes that normally catalyze the formation of peptide bonds for biotransformation. In peptide syntheses that make use of the 'reverse hydrolysis potential' of proteases several problems need to be considered, especially the necessity of minimizing competing hydrolysis of weakly activated acyl donor esters and the need to circumvent undesired product cleavage. Some approaches to suppress competitive reactions have been developed in our group, namely leaving group manipulations at the acyl donor in kinetically controlled reactions, enzymatic synthesis in organic solvent-free micro-aqueous systems, cryoenzymatic peptide synthesis, and biotransformations in frozen aqueous systems. Finally, for the first time, zymogens, which are known as catalytically inactive precursors of proteases, could be used as biocatalysts for practically irreversible peptide bond formation.

Biotransformation↗