Hans J. Vogel

Metal-ion-dependent hydrophobic-interaction chromatography of α-lactalbumins

α-Lactalbumins from bovine, human, goat, sheep, and horse milk bind to phenyl-Sepharose in the presence of EDTA and can be eluted by addition of Ca2+ (0.001–100 mm). This property has been utilized to purify these proteins in a one-step purification from milk whey. α-Lactalbumin purified in this manner has the same ultraviolet and proton nuclear magnetic resonance spectra as that purified by other methods. Using binding to phenyl-Sepharose as an assay, the conformation of bovine α-lactalbumin upon the addition of several metal ions that are known to interact with this protein was investigated. Lanthanides, Mn2+, Mg2+, and Cd2+ can substitute for Ca2+, whereas Zn2+, Al3+, and Co2+ cannot. Surprisingly, whereas lower concentrations of La3+, Mn2+, and Cd2+ (1 mm and less) caused elution from the hydrophobic support, higher concentrations (10 mm) were ineffective. These observations can be rationalized assuming the presence of two distinct metal-ion binding sites with different specificities.

Phosphorus-31 NMR studies of maltose and glucose metabolism in Streptococcus lactis

SummaryStreptococcus lactis ferments glucose in a homolactic fashion but a heterolactic fermentation pattern is observed when it is grown on maltose. Using in vivo phosphorus-31 and carbon-13 NMR studies of glucose-metabolizing cells we confirmed that fructosediphosphate (FDP) is the major glycolytic intermediate and that the production of lactate causes major changes both in the intra- and extracellular pH values. Starved cells contain mainly 3-phosphoglycerate (3-PGA) and some phosphoenolpyruvate (PEP). Metabolism of maltose also brings about major changes in pH, but it was unclear from the poorly resolved in vivo spectra if FDP was the main glycolytic intermediate present. This question was further studied by analyzing perchloric acid extracts by phosphorus-31 NMR. These studies showed that glucose-metabolizing cells have higher levels of FDP and lower levels of inorganic phosphate (Pi) than maltose-metabolizing cells. 3-PGA always remained present in the latter cells suggesting that these exist in a semi-starved state which is probably the reason for their heterolactic fermentation pattern. In the course of these studies we also examined the effects of the inhibitors 2-deoxyglucose, fluoride and iodoacetate. We could demonstrate that by judicious choice of carbon sources and inhibitors one could completely reduce the intracellular Pi pool. This suggests that one should be able to regulate the shift from heterolactic to homolactic fermentation, as Pi is considered to be the most potent inhibitor of pyruvate kinase in these cells.

An in vivo 31P NMR comparison of freely suspended and immobilized Catharanthus roseus plant cells

31P NMR spectra obtained for Catharanthus roseus plant cells entrapped in agarose or alginate were compared to those obtained for freely suspended cells. Oxygenated buffer was circulated through the NMR tube in all three instances. Essentially no differences were observed in the levels of the major metabolites: ATP, NAD(H), UDPG, cytoplasmic Pi, and sugar phosphates. Furthermore, the saturation of ATP by Mg2+, the energy status as determined from the ADP/ATP ratio, as well as the intracellular cytoplasmic and vacuolar pH, were not affected by the entrapment in the polymers. Since the cytoplasmic pH for freely suspended cells drops about 0.5 pH units upon shifting from aerobic to anaerobic conditions, these results indicate that the majority of the entrapped cells are properly oxygenated and that products such as CO2 can diffuse away. In addition, we found that the uptake rate of Pifrom the medium was lower in the polymer entrapped cells, and that the utilization rate of Pi from the vacuolar pool was also considerably reduced. However, the same pattern of Pi uptake, storage and utilization was observed in all cases. Thus, all the results obtained with the noninvasive 31P NMR technique suggest that the entrapment in agarose or alginate does not adversely affect cell metabolism since the phosphate metabolism and the cytoplasmic pH appear unaltered.

Carbon-13 and proton NMR studies of post-mortem metabolism in bovine muscles.

Proton and carbon-13 NMR was used to investigate post-mortem metabolism in bovine muscles at 26°C during the first 10h after slaughter. WALTZ-16 decoupling was used to eliminate the proton couplings in the (13)C spectra and the jump and return pulse sequence was used to suppress the water resonance in the (1)H-NMR experiments. With carbon-13 NMR the glycogen breakdown and the lactate development could be followed. This was compared with the lactate, creatine and phosphocreatine development as measured by proton NMR. The intracellular pH was estimated from the chemical shift of the abundant dipeptide, carnosine, as measured in the (1)H- and (13)C-NMR spectra. These were compared with similar measurements obtained earlier using phosphorus-31 NMR. The three independently determined pH profiles were in excellent agreement with one another, as well as with results obtained with the standard iodoacetate extraction method. In the course of these studies we observed that the post-mortem metabolism in cow and heifer was slow and that it took four more hours to complete compared to bull or young bull. After 10 h the pH was 5·9 in bull and 6·1 in cow. Phosphocreatine had completely disappeared after 3·5 h in bull samples while the lactate continued to increase even after 10h. The curves obtained by carbon-13 and proton NMR for the increase in lactate during the first 10 h post mortem were very similar. Moreover, plots for the increase in the lactate level versus the intracellular pH decrease showed a linear relationship, indicating that anaerobic glycolytic activity is the main determining cause for the intracellular pH decrease. Various other parameters, such as the ratio of unsatirated to saturated fatty acid side chains and the presence of amino acids and taurine, could be measured from the in vivo carbon-13 NMR spectra. However, no gross changes occured in any of these parameters during the first 10 h post mortem.

Post-mortem metabolism in fresh porcine, ovine and frozen bovine muscle

Post-mortem metabolism was followed by phosphorus-31-NMR in muscle samples obtained from freshly slaughtered pigs and lambs. Resonances for creatine phosphate (CP), ATP, inorganic phosphate (Pi) and sugar phosphates (SP) could be discerned and the intracellular pH could be determined from the spectra. The rates of post-mortem metabolism varied in the following fashion: porcine muscle > ovine muscle > bovine muscle. However, the course of post-mortem metabolism was, in all cases, the same. CP disappeared first and then ATP. Simultaneously, Pi increased, while SP remained relatively constant. The intracellular pH decreased to pH 5·5 in all tissues. In a separate set of experiments the post-mortem metabolism during thawing was studied in bovine muscles that had been frozen immediately after slaughter. Again, the same course of post-mortem metabolism was observed, but the thaw shortening was accompanied by an extremely rapid post-mortem metabolism, which was more than ten times as fast as that measured for fresh bovine muscles. The intracellular pH decreased from 7·2 to 5·5 in 45 min. This rapid metabolism started only after the sample ha reached 0°C. Resonances for metabolites were broadened in frozen muscles due to the limited motions that are allowed within the ice lattice.

Post-mortem energy metabolism in bovine muscles studied by non-invasive phosphorus-31 nuclear magnetic resonance

Phosphorus-31 Nuclear Magnetic Resonance ((31)P-NMR) has been utilized to follow non-invasively the post-mortem metabolism of the major phosphorylated metabolites in muscles from beef slaughter carcasses. In addition to adenosine-5-triphosphate (ATP), creatine phosphate (CP) and inorganic phosphate (P(i)) considerable amounts of glucose- and fructose-6-phosphate (G6P and F6P, respectively) as well as glycerol-3-phosphate (Glyc3P) were detected. ATP was mainly present as a Mg(2+)-ATP complex. Adenosine-5-diphosphate (ADP) appeared to be mainly bound to muscle proteins. A good quantitative agreement was found for the levels of ATP, CP and sugar phosphates (SP) when estimated by NMR or enzymatic assays. Since the chemical shifts of the P(i) and sugar phosphate resonances are a function of the pH, the intracellular pH could be directly deduced from the NMR spectra. Values obtained in this manner were, within the errors of both methods, the same as those determined in iodoacetate/KCl homogenates. The pH gradients within the tissue never exceeded 0.3 pH units. In a final set of experiments we used (31)P-NMR 10 study the effects of electrical stimulation on the intracellular pH and post-mortem metabolism. It was concluded that (31)P-NMR, due to its non-invasive nature plus the fact that some of the NMR parameters are sensitive to the intracellular environment, provides a useful complement to existing methods for the study of post-mortem metabolism.

31 P-nuclear magnetic resonance study of milk fractions

Milk serum, whey and milk ultrafiltrate were examined by 31 P nuclear magnetic resonance ( 31 P-NMR). About 20 phosphorylated milk constituents gave rise to resonances in the spectra. Most of these have been assigned to such well-known milk constituents as inorganic phosphate, N -acetylglucosamine-1-phosphate and glycerophosphorylcholine. Resonances from previously unknown constituents such as phosphocreatine were also observed. When the pH-dependence of inorganic phosphate, N -acetylglucosamine-1 -phosphate, glycerophosphorylcholine and gly-cerophosphorylethanolamine was examined it was observed that the resonance of inorganic phosphate overlapped that of N -acetylglucosamine-1-phosphate around neutral pH. This is the most probable explanation as to why this constituent was not observed in earlier 31 P-NMR studies on milk.

Trifluoperazine binding to calmodulin: A shift reagent 43Ca NMR study

43Ca NMR experiments of Ca2+ binding to calmodulin (CaM) were performed in the presence and absence of the calmodulin antagonist trifluoperazine (TFP). By making use of the shift reagent Dy(PPP)(7-) (a 1:2 complex of DyCl3 and Na5P3O10) we have succeeded in separating the 43Ca resonances of protein-bound Ca2+ and free Ca2+ in the otherwise unresolved spectra. This experimental strategy has allowed us to demonstrate unequivocally that the affinity of CaM for Ca2+ is markedly increased in the presence of TFP. Thus Ca2+ is not liberated from the protein upon addition of TFP as had been suggested based on earlier 43Ca NMR experiments (Shimuzu, T., Hatano, M., Nagao, S. and Nozawa, Y. (1982), Biochem. Biophys. Res. Comm. 106, 1112-1118).

Phosphorus-31 NMR studies of smooth muscle from guinea-pig taenia coli

Phosphorus-31 NMR spectra of superfused isometrically mounted guinea-pig taenia coli were obtained using a horizontal probe at 103.2 MHz. The spectra showed resonances for ATP, phosphocreatine (PCr), and a sugar phosphate resonance. The PCr/ATP ratio was between 1.5 and 2.0 consistent with chemical analysis of tissue extracts. The level of PCr, but not of ATP, decreases reversibly during contraction or inhibition of respiration. These conditions did not cause substantial changes in the intracellular pH, which was 7.0 ± 0.1.

Noninvasive 31P NMR Studies of the Metabolism of Suspended and Immobilized Plant Cells

Whole cell immobilization has become an important area of research. Large parts of the metabolism of the immobilized cells may be utilized for biosynthetic purposes. Simple, inexpensive carbon sources can be converted to very complex, valuable compounds. This is true in particular for immobilized plant cells producing secondary metabolites such as alkaloids and steroids. Preparations of immobilized whole cells have been characterized in various ways. However, noninvasive methods of studying the intact cell in the immobilized state have been lacking. In vivo P NMR has recently emerged as a very useful method for the study of bioenergetics and metabolism of living systems. Here we report on our comparative P NMR studies of freely suspended and immobilized plant cells. The technique is also applicable to other types of immobilized cells. Plant cells have been immobilized by entrapment in various matrices with viability and biosynthetic capacity pre~erved.~. Such immobilized cell preparations have been employed for de novo synthesis, for synthesis from metabolically distant precursors, or for bioconversions. These studies indicate that the secondary metabolism in immobilized plant cells is not largely altered by entrapment in the polymer. In this study, we have investigated several aspects of phosphate metabolism. In vivo studies of both free and immobilized cells of Catharanthus roseus and Daucus carota were performed. The cells were cultivated in submerged cultures in a modified growth medium (low concentration of manganese, which is paramagnetic and therefore can interfere in the NMR experiments), and they were entrapped in alginate or agarose according to standard procedures!*5 Phosphorus-3 I NMR has the advantage that the phosphorus nucleus has a spin of 1/2 and is 100% abundant, thus alleviating the need for expensive enrichment procedures. This, combined with the reasonable sensitivity of the IP nucleus for N M R experiments, has contributed to the recent popularity of P N M R studies. Only a few low-molecular-weight, phosphorus-containing compounds are present normally in the cells, so chemical shift assignments can usually be made readily. Long accumulation times are, however, often necessary to obtain spectra with a reasonable signal to noise ratio.