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K Jung

Publications and source records attributed to K Jung.

At least 19 recordsLinked to original sources
PubMed1995

Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli.

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coli lie in close proximity [Jung, K., Jung, H., Wu, J., Privé, G.G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. In this study, Glu269 was replaced with His in wild-type permease, leading to the presence of bis-His residues between helices VIII and X. Wild-type and Glu269-->His permease containing a biotin acceptor domain were purified by monomeric avidin affinity chromatography, and binding of Mn2+ was studied by electron paramagnetic resonance (EPR) spectroscopy. The amplitude of the Mn2+ EPR spectrum is reduced by the Glu269-->His mutant, while no change is observed in the presence of wild-type permease. The Glu269-->His mutant contains a single binding site for Mn2+ with a KD of about 43 microM, and Mn2+ binding is pH dependent with no binding at pH 5.0, stoichiometric binding at pH 7.5, and a midpoint at about pH 6.3. The results confirm the conclusion that helices VIII and X are closely opposed in the tertiary structure of lac permease and provide a novel approach for studying helix proximity, as well as solvent accessibility, in polytopic membrane proteins.

Binding Sites↗
PubMed1995

Role of glycine residues in the structure and function of lactose permease, an Escherichia coli membrane transport protein.

By using oligonucleotide-directed, site-specific mutagenesis, the role of 34 Gly residues in the lactose permease of Escherichia coli has been studied systematically. Each of 34 out of a total of 36 Gly residues was replaced with Cys in a functional permease mutant devoid of Cys residues (C-less permease), as previous experiments demonstrate that Gly-402 and Gly-404 can be deleted by truncation of the C-terminus with no loss of activity [Roepe, P. D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992; McKenna, E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969]. Out of the 34 Cys-replacement mutants described, 15 transport lactose with high activity, 16 exhibit decreased but significant ability to catalyze lactose accumulation, and 3 (Gly-64-->Cys, Gly-115-->Cys and Gly-147-->Cys) exhibit no activity whatsoever. The inactive mutants were studied in more detail by replacement of Gly with Ala, Val, or Pro. C-less permease with Gly-115-->Ala or Gly-147-->Ala transports lactose almost as well as the control, while mutants with Val or Pro in place of Gly have little or no capacity to accumulate the disaccharide. In contrast, mutants with Ala, Val, or Pro in place of Gly-64 are inactive. Strikingly, however, when the mutations are placed in the wild-type background, Gly-64-->Ala permease transports lactose, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside, and methyl 1-thio-beta,D-galactopyranoside 40-60% as well as wild-type permease, while Gly-64-->Val or Gly-64-->Pro permease is inactive toward all of these substrates. The results indicate that although none of the Gly residues in lactose permease is mandatory for activity, the bulk of the side chain at positions 64, 115, and 147, rather than conformational flexibility at these positions, is particularly important.

Amino Acid Sequence↗
PubMed1995

Lower serum high-density lipoprotein-cholesterol concentration in patients undergoing maintenance hemodialysis with acetate than with bicarbonate.

The authors studied the effect of dialysate buffer composition on the serum lipid values in male patients undergoing maintenance hemodialysis. Twenty patients on acetate and 21 patients on bicarbonate hemodialysis were investigated. The serum concentrations of high-density lipoprotein (HDL)-, HDL2- and HDL3-cholesterol were significantly lower in patients on acetate than in those on bicarbonate dialysis. The results of this study suggest possible adverse effects of acetate hemodialysis on serum HDL-cholesterol compared with bicarbonate hemodialysis. Other possibly contributory factors such as age and time on dialysis could be excluded. However, a possible role of differences in drug therapy between acetate- and bicarbonate-treated dialysis patients could not be entirely excluded.

Acetates↗
PubMed1995

Typing of coagulase-negative staphylococci from peritonitis in CAPD-patients by the PhP-CS system and REA.

Coagulase-negative staphylococci (CNS) were the most common bacteria causing peritonitis in patients treated with continuous ambulatory peritoneal dialysis (CAPD). In order to investigate if the same clone was responsible for the peritonitis in the different patients and if the exit site was the source of infection we followed 68 patients on CAPD for 2 years. During this period 9 patients had 12 episodes of peritonitis caused by CNS. Cultures were taken from exit site and peritoneal fluid in all patients at peritonitis and during the first study year at monthly intervals. In each culture up to 10 isolates of CNS were randomly collected and frozen. All 437 CNS isolates from the patients with CNS peritonitis were typed using a biochemical typing method and 41 isolates identical by this method were further discriminated by a DNA fingerprinting method. Identical strains were in no case isolated from different patients, indicating that no virulent strain was spread between the patients. The isolates causing the peritonitis were never found at the exist sites before the first day of the peritonitis in any patient. In only two patients was the same strain found at the exit site and in the peritoneal fluid on the first day of peritonitis. It thus seems that no virulent clone of CNS was infecting the patients and we found no evidence of CNS at the exit site causing the peritonitis.

Adult↗
PubMed1995

Effects of preservation solutions on cortical and medullary mitochondria of rat kidney.

The mitochondria isolated from cortex and medulla of rat kidneys were examined after storage in either Euro-Collins solution, in Bretschneider's histidine-tryptophan-ketoglutarat solution or in Belzer's University of Wisconsin solution at 25 degrees C or at 4 degrees C for a maximum of 4 to 24 hrs. Independently to the preservation of the solution used, the storage of the kidneys led to a decrease in state 3 respiration and uncoupled respiration as well as to an increase in the rate of state 4 respiration. The decrease in state 3 respiration ran parallelly to a decrease in adenine nucleotides. For the homogeneous protection of the mitochondria as well from the cortex as from the medulla, the Bretschneider's solution had the best preservative effect at a storage temperature of 25 degrees C as shown by the measured mitochondrial parameters. At 4 degrees C storage temperature, the differences in action with the various examined solutions were markedly smaller. However, compared to the other two solutions, the Euro-Collins solution showed a more favourable protective effect on cortical and medullary mitochondria.

Adenine Nucleotides↗
PubMed1994

Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site. 1. Site-directed mutagenesis studies.

Cys 148 in the lactose permease of Escherichia coli has been replaced with hydrophobic (Ala, Val, Ile, Phe), hydrophilic (Ser, Thr), or charged (Asp, Lys) residues, and the properties of the replacement mutants have been analyzed. Although Cys 148 is not essential for transport, the size and polarity of the side chain at this position modifies transport activity and substrate specificity. Thus, small hydrophobic side chains (Ala, Val) generally increase the apparent affinity of the permease for substrate, while hydrophilic side chains (Ser, Thr, Asp) decrease apparent affinity and bulky or positively charged side chains (Phe, Lys) virtually abolish activity. In addition, hydrophilic substitutions (Ser, Thr, Asp) alter the specificity of the permease toward monosaccharides relative to disaccharides. On the basis of these and other observations, it is concluded that Cys 148 is located in a sugar binding site of lac permease and probably interacts hydrophobically with the galactosyl moiety. The postulate receives more direct support from site-directed fluorescence labeling studies presented in the following paper in this issue [Wu, J., & Kaback, H. R. (1994) Biochemistry (following paper in this issue)].

Amino Acid Sequence↗
PubMed1994

Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling.

Recently we described the use of site-directed pyrene labeling of engineered lactose permease containing paired Cys residues to obtain proximity relationships between helices in the C-terminal half of the molecule [Jung, K., Jung, H., Wu, J., Privé, G. G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. Pyrene excimer fluorescence was detected for the double Cys mutants His322-->Cys/Glu325-->Cys, Arg302-->Cys/Glu325-->Cys, and Glu269-->Cys/His322-->Cys, indicating that helix X (His322-->Cys/Glu325-->Cys) is in an alpha-helical conformation and that helices VIII (Glu269-->Cys) and IX (Arg302-->Cys) are close to helix X (His322-->Cys and Glu325-->Cys). In this report, these interactions are used to study dynamic aspects of the permease. Excimer fluorescence between helices VIII and X or helices IX and X is markedly diminished by sodium dodecyl sulfate, while the excimer observed within helix X is unaffected, suggesting that tertiary interactions are disrupted by the denaturant with little effect on secondary structure. Furthermore, excimer fluorescence observed between helices VIII (Glu269-->Cys) and helix X (His322-->Cys) is quenched by Tl+, and the effect is markedly and specifically attenuated by ligands of the permease, suggesting that the pyrene becomes less accessible to the aqueous phase. The reactivity of single Cys residues at positions 269 or 322 was also examined by studying the rate of increase in fluorescence with N-(l-pyrenyl)maleimide. With both mutants, ligands of the permease cause a dramatic increase in reactivity which is consistent with the notion that these positions are transferred into a more hydrophobic environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine↗
PubMed1994

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.

Cysteine↗
PubMed1994

Evaluation of a microplate assay specific for heavy metal toxicity.

A rapid, quantitative microbial assay, which is specific for heavy metal toxicity, has been developed. The assay (MetPLATE) is in a 96-well microtitration plate format and is suitable for determining toxicity characteristics such as median inhibitory concentrations. The sensitivity of MetPLATE to heavy metals [Cu, Zn, Cd, Pb, Hg, Cr(III)] was generally higher than Microtox and was of the same order as or better than Daphnia and fish bioassay. MetPLATE was insensitive to organic compounds at concentrations higher than those found in the environment. Six out of 10 industrial wastewaters or process waters surveyed were toxic. Heavy metal analysis of these waters confirmed the presence of heavy metals in the toxic samples. MetPLATE can be run concurrently with other assays for general toxicity to help determine the nature of chemicals causing toxicity.

Animals↗
PubMed1994

Crotonobetaine reductase from Escherichia coli--a new inducible enzyme of anaerobic metabolization of L(-)-carnitine.

Crotonobetaine reductase from Escherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, gamma-butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, gamma-butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40-45 degrees C). The Km value for crotonobetaine was determined to be 1.1 x 10(-2M). gamma-Butyrobetaine, D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For gamma-butyrobetaine (Ki = 3 x 10(-5M)) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.

Anaerobiosis↗
PubMed1994

Nutrient intake of endurance runners with ovo-lacto-vegetarian diet and regular western diet.

During an endurance run (1,000 km in 20 days) it was investigated whether an ovo-lacto-vegetarian diet (OLVD) could cover the nutritional requirements of endurance athletes. A regular western diet (RWD) was used as reference. Both diets were offered with an energy content of 4,500 kcal per day and an energy percentage of carbohydrate:fat:protein of 60:30:10. The runners were divided into two dietary groups according to their usual dietary habits. The results of the 55 participants who completed the race show that runners from both groups had the same intake of energy, carbohydrate, fat and protein. Runners of the OLVD group consumed more dietary fiber and polyunsaturated fatty acids as well as less cholesterol. With the exception of sodium chloride and cobalamin, the intake of the calculated minerals and vitamins was higher in the OLVD and exceeded the official recommendations. This study shows that an OLVD with a high nutrient density is adequate to cover the nutritional requirements of endurance-athletes. The intake and absorption of iron should be monitored closely in all diet groups.

Adult↗
PubMed1994

Changed excretion of urinary proteins and enzymes by chronic exposure to lead.

Fifteen various serum and urine parameters were evaluated as indicators of renal alterations induced by lead in 82 male workers of a battery plant chronically exposed to lead (median of blood lead concentration: 2.03 mumol/l). The control group comprised 44 non-exposed healthy volunteers (0.34 mumol/l). High-molecular-mass proteins (transferrin, immunoglobulin G (IgG), (albumin)) were determined in urine as markers of glomerular integrity; low-molecular-weight proteins and parenchymal enzymes (alpha 1-microglobulin, beta 2-microglobulin, retinol-binding protein, lysozyme, ribonuclease, N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), alkaline phosphatase (AP), gamma-glutamyltransferase (GGT)) as indicators of changes in the proximal tubule; Tamm-Horsfall glycoprotein and kallikrein as markers of the distal tubule. There was a positive correlation between tubular indicators and blood lead concentration as well as the erythrocyte protoporphyrin (EPP). About 30% of the lead-exposed workers showed an increased excretion of alpha 1-microglobulin, NAG, ribonuclease, and/or Tamm-Horsfall protein, whereas the glomerular indicators remained unchanged. The combined determination of NAG and alpha 1-microglobulin in urine could be helpful in the early detection of lead-induced changes in the nephron.

Acetylglucosaminidase↗
PubMed1994

The lactose permease meets Frankenstein.

The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins. Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity. Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical. Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops. A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif. This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease.

Amino Acid Sequence↗
PubMed1994

Nephron target sites in chronic exposure to lead.

With established urinary markers of kidney integrity the early renal effects of lead have previously been considered to be mainly tubular or tubulointerstitial. In a cross-sectional study on 81 male lead-exposed workers and 45 age-matched controls (median blood lead concentrations 2.03 and 0.34 mumol/l respectively) not only well-established but also new urinary markers of renal integrity preferentially or exclusively located along the different nephron segments were analysed. Markers related to the glomerulus were 6-keto-prostaglandin 1 alpha, thromboxane B2, mainly produced in the glomerulus, and the extracellular matrix protein fibronectin. Markers of the proximal tubule were the brush-border antigens BBA, BB50, and HF5 and the intestinal alkaline phosphatase. Prostaglandin E2 and F2 alpha, preferentially synthesized in the collecting duct and medullary interstitial cells, served as markers of these more distal nephron segments. In contrast to previous studies on the early phase of lead nephrotoxicity, not only tubular but also glomerular involvement could be shown in the study presented here by increases in the median values of 6-keto-prostaglandin 1 alpha and decreases in fibronectin. The proximal tubular markers intestinal alkaline phosphatase and BBA confirmed that this particular segment of the nephron is affected by lead. Effects on the collecting tubule or medullary interstitial cells could also be observed. It is concluded that lead affects both the glomerulus and the tubular apparatus and that combinations of new and established markers could be valuable for a better definition and early detection of lead nephropathy.

Adult↗
PubMed1994

Urinary enzymes and low molecular weight proteins as markers of tubular dysfunction.

Reference intervals of different tubular markers, that is, low molecular weight proteins and urinary enzymes, show divergent data and wide ranges. The problems in establishing reference intervals for the tubular markers are caused by the necessarily different analytical methods. Also, the general rules of determining reference limits as well as the numerous physiological variables influencing tubular function are often not sufficiently taken into consideration. Compared to blood components, urinary tubular markers show a wide variability of values. This is due to the fact that the excretion of enzymes and proteins into urine represents an excretion into an open system. The influences of variables like age, sex, physical exercise, different urine flow rates, and biorhythms are immediately reflected by changed excretion rates of tubular markers. The problems occurring when the second morning urine sample is being used as a "standardized" collection method and the basis to characterize tubular function by analyte/creatinine ratios are discussed in this paper.

Adolescent↗
PubMed1994

Early urinary markers of target nephron segments as studied in cadmium toxicity.

A number of chemicals may adversely affect one or more of the anatomical structures of the kidney, such as the glomerulus, the tubular apparatus, the medullary, or interstitial cells. To recognize subclinical renal dysfunction, a battery of new, non-invasive tests was applied in comparison to established ones. The study on cadmium exposed subjects, performed within the framework of a collaborative European research project, exemplifies the concept of target selectivity within a nephron. One hundred seventy-two subjects were classified according to urinary cadmium excretion as controls (< 1.5 micrograms/g creatinine), or subjects with moderate or high cadmium body burden (1.5 to 5 micrograms/g creatinine, > 5 micrograms/g creatinine). Twenty-six urinary analytes (such as serum derived proteins, tubular enzymes, eicosanoids) and four plasma markers, related to the function or integrity of specific nephron segments, were investigated in a cross-sectional study. The group with the moderate cadmium body burden showed alterations of proximal tubular integrity, that is, increased excretion of tubular brush-border antigens. The group with higher cadmium body burden revealed an involvement of the whole nephron. The most prominent quantitative changes were found for the glomerular markers high molecular weight proteins, and thromboxane B2 and for the proximal tubular markers retinol binding protein, alpha 1-microglobulin, N-acetyl-beta-D-glucosaminidase, and the intestinal alkaline phosphatase. A diagnostic approach to screen for nephrotoxicity due to environmental hazards like cadmium should include proximal tubular markers (alpha 1-microglobulin and tubular enzymes, that is, intestinal alkaline phosphatase) but the measurement of glomerular markers is also advisable.

Adult↗