Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

Human complex-forming glycoprotein, heterogeneous in charge: the primary structure around the cysteine residues and characterization of a disulfide bridge

L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity (v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of $\text{1}\text{v}$ versus $\text{1}\text{ATP}$. Michaelis constants of the two enzymes were calculated to be 22 and 173 μm, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μm ATP compared to 1 μm ATP. Further studies of the low K_m (22 μm) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg²⁺, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low K_m enzyme.     

Photoactivated cross-linking of prolactin to hepatic membrane binding sites

The oxidized chemotactic peptide N-formyl-L-methionyl-sulfoxide-L-leucyl-L-phenylalanine can be reduced using either a partially purified methionine sulfoxide peptide reductase from $\text{Escherichia}\text{coli}$ or a neutrophil extract. The product of the enzymatic reduction shows chemotactic activity.     

Effects of dimethyl sulfoxide and glycerol on catalytic and regulatory properties of glutamine-dependent carbamoyl phosphate synthase from rat liver and dual effects of uridine triphosphate.

The cryoprotectants dimethyl sulfoxide and glycerol markedly affected the activity of glutamine-dependent carbamoyl phosphate synthase (EC 2.7.2.9) of rat liver, the first enzyme of de novo pyrimidine biosynthesis. The apparent K_m for MgATP^2? decreased with increases in the solvent concentrations, from 7.0 mm without the solvents to 0.1 and 0.8 mm in the presence of 25% ($\text{v}\text{v}$) dimethyl sulfoxide and 30% ($\text{w}\text{w}$) glycerol, respectively. The apparent K_m for bicarbonate also decreased with these solvents, while that for glutamine was not significantly affected. The response in the maximal velocity to the solvents was biphasic; the value of V increased 1.39- and 1.18-fold with 7.5% dimethyl sulfoxide and 10% glycerol, respectively, but then decreased as the concentrations of the solvents were further increased. The extents of inhibition by 25% dimethyl sulfoxide and 30% glycerol were 63 and 44%, respectively. The effects of 5-phosphoribosyl 1-pyrophosphate and MgUTP, allosteric effectors, were greatly modified by these solvents. Kinetic parameters as affected by the effectors in the presence of various concentrations of the solvents are described. A notable observation was that MgUTP, a potent inhibitor under ordinary conditions, stimulated the activity in the presence of high concentrations of dimethyl sulfoxide; in the presence of 30% dimethyl sulfoxide and 1 mm MgATP^2?, 0.5 to 2.0 mm MgUTP enhanced the enzymatic activity about twofold. MgUTP at higher concentrations was inhibitory. The dimethyl sulfoxide-dependent dual effects of MgUTP indicate the possible existence of at least two MgUTP-binding sites on the enzyme molecule.     

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

One hour following administration of physiological concentrations of the steroid hormone antheridiol to a male strain of the water mold, $\text{Achlya}$$\text{ambisexualis}$, the rate of total cellular protein synthesis is increased. Further analysis revealed a sequential increase in the rate of syntheses for three classes of proteins following hormone stimulation. The rate of ribosomal protein synthesis increased as early as 20–30 minutes, followed by ribosomal salt wash proteins (40–60 minutes) and total soluble proteins after 60 minutes. Patterns of total cellular proteins, resolved by two-dimensional gel electrophoresis, during the first four hours after hormone treatment demonstrated the appearance of two newly synthesized peptides beginning at approximately 40 minutes followed by an increased rate of synthesis of three peptides after one hour. The synthesis of two peptides totally decreased after three hours of hormone induction.     

Peptide purification by gel filtration in aqueous pyridine--ammonia.

A method is described for the purification of peptides by gel filtration on Sephadex. The success of the method is due mainly to the use of 70% ($\text{v}\text{v}$) pyridine-30% ($\text{v}\text{v}$) 1 m aqueous ammonia, an excellent volatile solvent for peptides which does not degrade Sephadex. The method has been used to purify all of the major peptides of cowpea chlorotic mottle virus coat protein, after an initial fractionation by ion-exchange chromatography, and selected separations are used to illustrate the degree of fractionation which can be achieved.     

Subzero preservation of mechanically prepared porcine islets of Langerhans: Response to a glucose challenge in vitro

Islets of Langerhans, prepared by a new mechanical process and avoiding enzymatic digestion were frozen to ?196 °C. Two rates of freezing were compared, instantaneous directly into liquid nitrogen and slow freezing at 1 °C min^?1. Post-thaw survival was greater after slow freezing.Three concentrations of dimethyl sulfoxide (DMSO) were compared. The 10% $\text{v}\text{v}$ concentration was found to give greater success than 20 or 30%. Contaminating exocrine tissue was found not to survive the freezing process.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.     

Extraction of pure ribosomal protein and removal of Coomassie blue from acrylamide gels

A method has been developed to recover pure ribosomal proteins from two-dimensional polyacrylamide electrophoresis slabs. Proteins and Coomassie blue were extracted from the gels and separated by passing them through a Sephadex G-25 column. All the steps were performed in the presence of 66% ($\text{v}\text{v}$) acetic acid. The technique has been applied to Escherichia coll ribosomal proteins labeled with ¹²⁵I.     

Effects of cytochalasin B on division and calcium carbonate extrusion in a calcareous alga.

Cytochalasin B (CB) (100 μg/ml) reversibly blocked cell division and cuased the formation of abnormal cytoplasmic bodies in the alga Cricosphaera carterae. Concentrations of 20 μg/ml and 40 μg/ml CB were without effects. In the presence of CB, calcified bodies (coccoliths) which form in Golgi vesicles and are normally extruded through the plasma membrane were not extruded and accumulated within the cell. CB appeared to alter the membranes of Golgi vesicles containing coccoliths. DMSO (10% $\text{v}\text{v}$), the solvent for CB, was without effect on cell division and coccolith extrusion. A concentration of 20% $\text{v}\text{v}$ DMSO inhibited cell division irreversibly.     

Stabilization of proteins immobilized on Sepharose from leakage by glutaraldehyde crosslinking

A technique has been developed to prevent leakage of proteins immobilized on Sepharose without destroying their biological functions. This involves the use of glutaraldehyde at concentrations ranging from 0.015 to 0.25% ($\text{v}\text{v}$) to crosslink proteins, which had been coupled to Sepharose by conventional methods. Glutaraldehyde crosslinking decreases immuno-globulin G leakage from Sepharose-immunoadsorbents to undetectable levels without noticeably affecting antigen-binding activity and reduces leakage of lactoperoxidase from solid-phase lactoperoxidase with only a moderate reduction of enzymatic activity.     

Modified colorimetric ninhydrin methods for peptidase assay

Four colorimetric procedures suitable for the determination of peptidase activity on peptides having a free α- or ε-amino group are described. Two of the methods (A and B) are modifications of the conventional ninhydrin method described by S. Moore and W. H. Stein ((1948) J. Biol. Chem.176, 367–388; (1954) Ibid.211, 907–913); the heating time is shortened to 5 min at 100°C and the pH of the buffer in the reagent is lowered to 4.0. Method A differs from method B in buffer concentration. The other two methods (C and D) are modifications of the Cd-ninhydrin method described by A. P. Tsarichenko ((1966) Nauch. Tr. Krasnodar. Gos. Pedagog. Inst.70, 86–88, as cited in Chem. Abs.67, 79479c); the water content in the reagent is reduced to $\text{1}\text{20}$ of the original reagent and the sample is heated for 5 min at 84°C. Method C differs from method D in the ratio of sample to reagent. In contrast to the free amino acids which are sufficiently colored, various peptides and amino acid derivatives except for the glycylpeptides give only a faint color with these methods. These four methods are not only useful for the determination of peptidase activity on peptides (e.g., Leu-Gly and tert-butyloxycarbonyl-glycyl-lysyl-leucine), but are also useful for the determinations of amidase activity on amino acid amides (e.g., Leu-NH₃) and esterase activity on amino acid esters (e.g., tyrosine ethyl ester).     

Interrelations between pH and temperature for the catalytic rate of the M4 lsozyme of lactate dehydrogenase (EC 1.1.1.27) from goldfish (Carassius auratus L.).

A kinetic study of the rate of pyruvate reduction by goldfish LDH-M₄ (the homotetrameric form of lactate dehydrogenase which predominates in skeletal muscle) provided an analysis of the effects of pH and temperature on v (reaction velocity) and estimates of how temperature might affect catalysis in vivo, where the physiological pH regulation imposes a temperature coefficient of ?0.015 to ?0.020 pH unit/ °C. Consistent with published data for other LDHs, (i) V (maximum reaction velocity) was pH insensitive over a physiological pH range, (ii) the K_m for pyruvate, K_P, was sensitive to both pH and temperature, and (iii) the K_m for NADH and the dissociation constant for NADH were both sensitive to temperature, but not to pH. V approximately doubled with each 10 °C (E_a = 11.7 kcal/mol). The effects of pH and temperature on K_P were consistent with two enthalpic contributions, an ionization enthalpy (ΔH_i^°) of 7.2 kcal/mol (probably a histidine imidazole), and an enthalpy (ΔH_SO) of 5.8 kcal/mol for the combination of pyruvate with the nonionized (pH ? pK′) LDH-NADH complex. When the pH was varied according to the physiological temperature coefficient, v was more sensitive to temperature than for conditions of constant pH, the usual design of kinetic experiments. This effect was due to the decreased temperature sensitivity of K_P caused by partial concellation of the ΔH_i^° effect by the pH regulation: $\text{d}\text{pH}\text{dT}\text{?}\text{d}\text{p}\text{K′}\text{dT}$. At constant pH, on the other hand, K_P increased strongly with temperature and had the effect of offsetting (especially at higher pH values) the large increases in V. It was suggested that the magnitudes of ΔH_i^° and ΔH_SO might have been important in the evolution of LDHs and other enzymes of cold-blooded animals.     

Control of mitochondrial respiration: a quantitative evaluation of the roles of cytochrome c and oxygen.

The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c has been measured as a function of the exogenous $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio and pH. The respiratory rate at $\text{[}\text{ADP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ values of less than 10^-1m^-1 is proportional to the reduction of cytochrome c and independent of pH from pH 6.5 to pH 8.O. The maximal turnover number (at 100% reduction) for cytochrome c is approximately 70 s^?1. As the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio is increased from 10^?1m^?1 to 10⁴m^?1, the respiration at any given level of reduction of cytochrome c is progressively inhibited. Greater inhibition is observed at more oxidized levels of cytochorme c with respiratory control values for oxidation of reduced cytochrome c exceeding 10. The behavior of mitochondrial respiratory control is shown to be quantitatively consistent with a proposed mechanism in which the regulation occurs in the reaction of oxygen with cytochrome oxidase. A steady-state rate expression is derived which fits the mitochondrial respiratory rate dependence on (i) the extramitochondrial $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio; (ii) the level of reduction of cytochrome c (or the intramitochondrial $\text{[}\text{NAD}{}^{\mathrm{+}}\text{]}\text{[NADH])}$ at different $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ values; (iii) the pH of the suspending medium. This rate expression appears to correctly predict the relationships of the cytoplasmic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio, the mitochondrial $\text{[}\text{NAD}{}^{\mathrm{+}}\text{]}\text{[}\text{NADH}\text{]}$ ratio, and the mitochondrial respiratory rate in intact cells as well as suspensions of isolated mitochondria.     

Irreversible inhibitors of methotrexate transport in L1210 cells: Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate

Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37°C, inhibition was rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 1}\text{min}$), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4°C. Inactivation in this case was qualitatively similar to the corresponding process at 37°C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4°C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.     

The mechanism of anion inhibition of pork heart aspartate aminotransferase

The mechanism of anion inhibition of the reaction of the pork heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1) with erythro-β-hydroxy-l-aspartate was investigated. This reaction produces a mixture of complexes, one of which is characterized by an absorption maximum at 492 nm. Spectrophotometric analysis of equilibrium mixtures of aspartate aminotransferase and erythro-β-hydroxy-l-aspartate, in different buffers, indicated that acetate, chloride, and cacodylate were competitive inhibitors of hydroxyaspartate binding. Pyrophosphate, however, was not a competitive inhibitor. Between pH 4.5 and 9.0 the affinity of the enzyme for the monovalent anions decreased as the pH increased. The data indicated that the anion binding group had a pK_a in the range from pH 6 to 7, depending upon the anion studied. From pH 4.5 to 9.0, the substrate dissociation constant and the distribution of enzyme-substrate complexes were both unaffected by pH. By stopped-flow spectrophotometry, an initial rapid relaxation ($\text{t}\text{1}\text{2}\text{= 2–8}\text{ms}$) was associated with an increase in absorbance at 492 nm, and this rate depended upon both substrate and buffer concentrations. A slower relaxation ($\text{t}\text{1}\text{2}\text{= 180}\text{ms}$) was associated with a decrease in the absorbance at 492 nm to approximately 70% of the value attained in the first rapid reaction. The rate of this slower reaction was largely independent of substrate and buffer concentrations. Kinetic analysis of the rates of the first relaxation in several different concentrations of Tris-acetate buffer of pH 8 showed that the rate of association decreased with increasing acetate concentration whereas the reaction rate for dissociation was unaffected. Thus, acetate appears to exert its inhibitory effect by preventing the formation of the enzyme-substrate complex rather than by displacing the substrate from the enzyme.     

Amino acid sequence of the heme a subunit of bovine heart cytochrome oxidase and sequence homology with hemoglobin.

The amino acid sequence of the 11.6 K dalton heme $\text{a}$ subunit of bovine heart cytochrome oxidase has been completed and is presented here. The sequence investigation has established the positions in the protein of all the possible heme ligands, namely cysteine, methionine, histidine and lysine residues. However, the isolation conditions may have caused the heme $\text{a}$ to migrate from its original site or the heme is caged by peptides as pointed out in Reference 6. The sequence of the heme $\text{a}$ subunit and the β-chain of hemoglobin shows homology. It is possible that these two proteins have arisen from a common ancestor in the distant past.     

Reduction of N-acetyl methionine sulfoxide in plants

An enzymic activity which catalyzes the reduction of N-acetyl-methionine sulfoxide to l-N-acetyl-methionine has been observed in a wide variety of plant tissues. Its activity depended on the presence of dithiotreithol in the incubation medium. l-Methionine-sulfoxide was essentially inactive as a substrate. Of all the physiological reductants tested, only thioredoxin partially replaced dithiothreithol. When fractions obtained by gradient centrifugation of gently disrupted barley protoplasts were assayed for the reductase, the activity was largely associated with chloroplasts although approximately 15% was found in the cytosolic compartment. The enzyme, isolated from spinach chloroplasts, had a broad pH optima between 7.0 and 8.0, and its K_m for N-acetyl methionine sulfoxide is 0.4 millimolar. The possible participation of this ubiquitous enzyme in enzyme regulation is discussed.