Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.     

Effect of Sodium Chloride and pH on Enterotoxin C Production

Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 10(8) cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 10(8) cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 10(8) cells per ml and 6.38 to 7.30 for 3.6 x 10(6) cells per ml. In repeated experiments in which the inoculum contained 10(8) cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts.     

Metabolism of D- and L-glyceraldehyde in adipose tissue: a stereochemical probe for glycerokinase activity

Distributions of (14)C have been determined in free glycerol, in glycerol from triglycerides, in glucose from glycogen, and in lactate after incubation of d-glyceraldehyde-3-(14)C and l-glyceraldehyde-3-(14)C with rat adipose tissue. The distributions are interpreted in terms of presently accepted possible reactions for the initial metabolism of glyceraldehyde. Formation of glycerol-1-(14)C from d-glyceraldehyde-3-(14)C indicates that in adipose tissue glyceraldehyde is reduced to glycerol. Incorporation of (14)C from d-glyceraldehyde-3-(14)C into carbon 3 of the glycerol of triglyceride indicates that d-glyceraldehyde is either phosphorylated or oxidized to d-glyceric acid, or both, in its initial metabolism. Incorporation of (14)C from l-glyceraldehyde-3-(14)C into carbon 3 of glycerol indicates that l-glyceraldehyde is reduced to glycerol, which is phosphorylated and (or) converted to d-glyceric acid via l-glyceric acid. Some (14)C from l-glyceraldehyde-3-(14)C is incorporated into carbon 1 of glycerol of triglycerides and carbon 4 of glycogen; the explanation for this incorporation is uncertain.     

Studies on the existence of a pathway in liver and muscle for the conversion of glucose into glycogen without glucose 6-phosphate as an intermediate

Mixtures of (14)C-labelled glucose plus pyruvate were incubated either with rat diaphragm or slices of rat liver. Incorporation of glucose carbon into glycogen was compared with its incorporation into glucose 6-phosphate relative to the incorporation of pyruvate carbon into these metabolic products. There was no preferential incorporation of glucose carbon relative to pyruvate carbon into glycogen compared with glucose 6-phosphate in the liver slices, but there was in diaphragm. On the assumption that glucose 6-phosphate is a necessary intermediate in the conversion of pyruvate carbon into glycogen, this is evidence for the existence in muscle, but not in liver, of more than one pool of glucose 6-phosphate or of a pathway from glucose to glycogen without glucose 6-phosphate as an intermediate. Galactose carbon, relative to pyruvate carbon, was preferentially incorporated into liver glycogen, so that a substrate converted in liver into glycogen without glucose 6-phosphate as an intermediate could be detected by this approach.     

Prophylactic Effects of γ-Aminobutyrylhistidine (Homocarnosine) on Experimental Staphylococcal Infections in Mice

Prophylactic administration of the dipeptide homocarnosine induced a high degree of resistance to staphylococcal infections in Swiss albino mice. It expressed its antistaphylococcal properties 1 hr after administration, and this protection lasted for at least 1 month. Although 5 mg per animal (approximately 200 to 250 mg/kg) was routinely used in our studies, experiments showed that comparable results could be obtained with 1.5 mg per animal. Rechallenge experiments indicated that an active infection by itself may confer immunity up to 4 weeks, but an infection after treatment with homocarnosine gave complete immunity to reinfection for at least 2 months. Studies in vitro showed that homocarnosine had no effect on the growth or certain other characteristics (ability to ferment mannitol, liquefy gelatin, and to produce coagulase, deoxyribonuclease, and pigment) of S. aureus. It appears that resistance induced by this peptide is an indirect effect mediated by some nonimmunological host reaction. The possible involvement of homocarnosine, among other compounds, in the protective action of deproteinized beef extract against staphylococcal infections is suggested.     

ACTION OF TRITON X-100 ON CHLOROPLAST MEMBRANES : Mechanisms of Structural and Functional Disruption

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100–200 µM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.