Ligation of fibrinogen by factor XIIIa with dithiothreitol: mechanical properties of ligated fibrinogen gels

Human fibrinogen (concentration 8.4 mg/mL) was ligated (cross-linked) with factor XIIIa and dithiothreitol (DTT) at pH 8.5, ionic strength 0.45. With 7.5 μg/mL of factor XIIIa alone, there was almost no γ-γ ligation, but with 2 mM DTT added, oligomers appeared, and γ-γ and Aα-Aα ligation was nearly complete after 3 days. At 38 μg/mL of factor XIIIa, some γ-γ and Aα-Aα ligation occurred even without DTT. For fibrinogen concentrations of 4.0 and 8.4 mg/mL, 38 μ/mL factor XIIIa, 2.0 mM DTT, clot-like gels formed and the shear modulus of elasticity increased slowly over several days to a constant value. The final modulus was similar in magnitude to those of ligated clots of α-fibrin (clotted by thrombin) and α-fibrin (clotted by batroxobin) under the same conditions. However, the opacity was somewhat higher; whereas in fine fibrin clots there is minimal lateral association of the protofibrils, in fibrinogen gels at the same pH and ionic strength the protofibrils (which are presumably single chains of fibrinogen monomers joined end to end at their D domains) are evidently associated in bundles (although not to the degree seen in coarse fibrin clots). Creep and creep recovery measurements showed almost perfect elastic behavior, with essentially no creep under stress and complete recovery after removal of stress. The modulus was scarcely affected by introduction of lithium bromide by diffusion to a concentration of 0.6M, which in unligated fibrin clots causes substantial softening. Whereas in fine fibrin clots (both αβ-fibrin and α-fibrin) factor XIIIa causes only γ-γ ligation, addition of 2 mM DTT produced some α-α ligation in these also.     

Interaction of fibrinogen-binding tetrapeptides with fibrin oligomers and fine fibrin clots

The polymerization of fibrin, at pH 8.5 and ionic strength 0.45, and under conditions where the action of thrombin on fibrinogen was the rate-determining step, was interrupted by inactivating thrombin with p-nitrophenyl-p′-guanidinobenzoate (NPGB). Addition of the tetrapeptide Gly-Pro-Arg-Pro (GPRP) partially dissociated the fibrin oligomers as shown by subsequent ligation with Factor XIIIa and calcium ion followed by denaturation and gel electrophoresis; polyacrylamide gel electrophoresis with reduction showed a decrease in the proportion of γ-γ ligation compared with controls untreated by GPRP, and agarose gel electrophoresis showed a shift in the distribution of oligomer sizes. The dissociation was accomplished within 15 min and its extent was consistent with establishment of an equilibrium in which two molecules of GPRP react to sever an oligomer. When GPRP was introduced into fine unligated fibrin clots by diffusion, there was some dissociation as shown by differences in the degree of γ-γ ligation after treatment by Factor XIIIa; but the action of GPRP was much slower and less complete than on soluble oligomers. However, even a small amount of dissociation affected the mechanical properties of fine clots profoundly. The shear modulus (measured 25 s after application of stress) decreased progressively with increasing concentration of GPRP introduced by diffusion. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. If the steadystate creep rate is interpreted in terms of an effective viscosity, the latter is decreased by up to three orders of magnitude by the presence of GPRP. In terms of transient network theories of viscoelasticity, the average lifetime of a network strand is greatly diminished. However, the total density of strands remains constant during creep and creep recovery as shown by constancy of the differential modulus or compliance. Removal of GPRP by diffusion only partially restores the original shear modulus and creep behavior of the original clot. Some limited data on the effect of the tetrapeptide Gly-His-Arg-Pro are also reported.     

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rheological studies of creep and creep recovery of unligated fibrin clots: comparison of clots prepared with thrombin and ancrod

Mechanical creep and creep recovery in small shearing deformations have been studied in unligated clots formed with both thrombin and ancrod. In thrombin clots, both A binding sites (which interact with “a” sites to link monomer units within a protofibril) and B sites (which interact with “b” sites to form links between protofibrils) are exposed to enable formation of linkages; in ancrod clots, only the A sites are exposed. Fine clots (with minimal lateral aggregation of protofibrils), coarse clots (with substantial aggregation of fibril bundles), and clots of intermediate coarseness were compared. Fine thrombin clots showed less creep at short times but more creep at long times than coarse or intermediate clots and had more irrecoverable deformation relative to the initial elastic deformation. Ancrod clots had greater irrecoverable deformation than the corresponding thrombin clots, both fine and coarse. The permanent deformation in fine ancrod clots was enormous, corresponding almost to fluid character; the rate of permanent deformation was larger than that in fine thrombin clots by more than two orders of magnitude. For all types of clots, differential measurements of compliance (or its reciprocal, elastic modulus), as well as the applicability of the Boltzmann superposition principle to calculation of creep recovery, showed that the overall density of structure remained constant throughout the mechanical history; i.e., if structural elements were breaking, they were reforming at the same rate in different configurations. The possibility that the weakness of ancrod clots is attributable to partial degradation of α-chains rather than absence of Bb linkages was eliminated by comparisons of clots made with thrombin, ancrod, and ancrod plus thrombin; the last two showed identical partial degradation of α-chains (by gel electrophoresis), but the first and third had essentially identical initial elastic moduli and creep behavior. Two alternative mechanisms for irrecoverable deformation in fine clots are discussed, involving rupture of protofibrils and slippage of twisted segments, respectively.     

Rheology of fibrin clots: III. Shear creep and creep recovery of fine ligated and coarse unligated clots

Creep and creep recovery of human fibrin clots in small shearing deformations have been investigated over a time scale from 24 to 10⁴ s. Coarse, unligated dots and fine dots ligated by fibrinoligase in the presence of calcium ions were studied to suppllement previous data on coarse ligated and fine unligated clots. Stress was found to be proportional to strain up to at least a maximum shear strain (in torsion geometry) of 2.6%. The initial modulus (25 s after imposition of stress) is proportional to approximately the 1.5 power of concentration for fine ligated and coarse unligated clots. For fine unligated clots, there is comparatively little creep subsequent to the initial deformation; ligation (in this case involving mostly the γ chains) reduces the creep to nearly zero. For coarse unligated dots, there is substantially more creep under constant stress, and creep recovery is not complete. legation (in this casa involving both γ and α chains) largely suppresses the creep and causes the recovery to be complete. If the structure is fully formed before creep begins, tests of creep recovery by the Boltzmann superposition principle show adherence to linear viscoelastic behavior for all four clot types. Otherwise, the Boltzmann test fails and the recovery is much less than calculated. For fine ligated clots, the observed recovery agrees well with that calculated on the basis of a dual structure model in which an additional independent structure is built up in the deformed state, so that the state of ease after removal of stress is a balance between two structures deformed in opposite senses, it is postulated that the coherence and elastic modulus of the fine ligated dot are largely due to steric blocking of long protofibrils with a high flexural stiffness. In the coarse clot, it is proposed that the structure involves extensive branching of thick bundles of protofibrils, which become permanently secured by the ligation of the α chains of the fibrin.     

Rheology of fibrin clots. v. shear modulus,creep, and creep recovery of fine unligated clots

Creep and creep recovery in small shearing deformations have been studied in fibrin clots at pH 8.5 and ionic strength 0.45, where the fine, transparent clot is formed with very little lateral aggregation of protofibrils. The initial shear modulus G₁ was measured 25 s after deformation on clots aged long enough for complete development of structure. For both human and bovine fibrin, the data were approximately described by log G₁ = 1.45 + 1.90 log c, where c is concentration in $\text{g}\text{l}$ and G₁ is in $\text{dyn}\text{cm}{}^2$, over a range of c from 4 to 13 $\text{g}\text{l}$. For bovine clots with completely developed structure, creep and creep recovery showed substantial irrecoverable deformation but the differential modulus G_Δ measured at intervals agreed with G₁ and did not change during the course of the experiment; it also agreed with the value calculated from the initial recovery after removal of stress. Moreover, several tests showed that the course of recovery conformed closely to the Boltzmann superposition principle. Thus the irrecoverable strain was associated with a structural rearrangement which caused no permanent damage. The irrecoverable deformation relative to the initial deformation was proportional to the elapsed time during creep in the early stages with a proportionality constant that decreased somewhat with increasing clot age prior to imposition of stress; it corresponded to a pseudo-viscosity of the order of 10⁷ poise. However, the irrecoverable deformation does not represent viscous flow and appears to approach a limiting value at long times. Experiments on clots without completely developed structure, i.e., with imposition of stress at an earlier clot age, showed an increase in the differential modulus G_Δ during creep. The irrecoverable deformation was greater and a portion of it could be attributed to the balance between two structures formed in the unstrained and strained states. However, unlike the case of ligated clots strained before complete development of structure, where the irrecoverable deformation is entirely due to a two-structure balance, there is also a contribution from structural rearrangement. Experiments with reverse creep and creep recovery showed that the structural rearrangement is symmetrical with respect to direction of deformation. The interpretation of these results in terms of clot structure and internal motions of protofibrils is discussed.     

Stress relaxation in fine fibrin films: Comparison of films prepared with thrombin and ancrod

Measurements of stress relaxation in uniaxial extension have been made on fibrin film prepared from fine bovine fibrin clots (i.e., clots in which there is minimal lateral aggregation of protofibrils), both ligated and unligated, and polymerized with both thrombin and ancrod, plasticized with either aqueous buffer or glycerol. The stress 100 s after imposition of strain was approximately proportional to In λ, where λ is the stretch ratio. Ligated thrombin films showed comparatively little relaxation over a period of one day and almost complete recovery after release of stress. In unligated thrombin films, there was substantial relaxation in two stages, as previously observed for coarse films, and substantial irrecoverable deformation. The extent of relaxation and the proportion of strain that was irrecoverable increased with the magnitude of the strain. In ancrod films (unligated), there was much more relaxation (stress decaying by as much as a factor of 10) and much more irrecoverable deformation (about 70% of the initial deformation); these results did not depend on the magnitude of the strain. When an ancrod film was released after relaxation and submitted to a second stretch, the extent of the second relaxation was much less. These observations are discussed in relation to the structure of fine films and possible mechanisms for relaxation and irrecoverable deformation.     

Protofibrin clots induced by calcium and zinc

During the course of studies with fibrin protofibrils, produced by adding hirudin to thrombin-activated fibrinogen prior to the onset of gelation, turbid clots were observed to be generated merely by adding Ca(II) or Zn(II) to protofibrils. The rate of gelation (CT) and turbidity of the “protofibrin” clots increases with cation levels in a concentration-dependent manner, with Zn(II) much more potent than Ca(II). For example, 50 μM Zn(II) generated a more turbid protofibrin clot than 0.5 mM Ca(II). In combination, levels of Zn(II) and Ca(II), which individually have no effect, induce protofibril gelation. The generation of protofibrin clots by Zn(II) is decreased at increasing ionic strength. Apparently, the underlying electrostatic forces that bind the monomers in fibrin and protofibrin gels are similar. SEM micrographs show that Ca(II)- or Zn(II)-induced protofibrin clots (600–1500Å thick) are essentially indistinguishable from those formed directly from fibrinogen and thrombin with divalent cation. The protofibrin fibers induced by the cations are thicker than the fibers formed directly from fibrinogen and thrombin in the absence of divalent cation. Branching appears brought about the the divalent cation-sensitive lateral association of different protofibril strands. These findings describe simple experimental methods for separately studying the early and late stages of fibrin gelation.     

Calcium-activated proteolysis of neurofilament proteins in goldfish Mauthner axons

We have examined the proteolytic breakdown of neurofilament proteins (NFPs) in isolated Mauthner axoplasm (M-axoplasm). Documentation of proteolytic breakdown of NFPs in M-axoplasm is important because NFPs are not degraded in distal segments of severed Mauthner axons (M-axons) maintained in vivo for up to 62 days at 20°C. By incubating M-axoplasm with 2 mM calcium in vitro, we have demonstrated that M-axoplasm contains an endogenous calcium-activated neutral protease that degrades NFPs. This calcium-activated proteolysis of M-axoplasm NFPs produced novel bands on silver-stained gels. These novel bands were presumed to be NFP breakdown products because they reacted with antibodies to the α-intermediate filament antigen (anti-IFA) on immunoblots from these gels. Incubations of M-axoplasm with 2 mM calcium plus exogenous calpain produced novel bands similar to those observed for M-axoplasm incubated with 2 mM calcium. Incubations of M-axoplasm with 2m M calcium plus calpain inhibitors did not produce these novel bands. These in vitro data indicate that M-axoplasm contains calpain that degrades NFPs and produces novel bands similar to those observed from distal segments of severed M-axons maintained in vivo longer than 62 days postseverance. Factors that affect the activity of calpain or affect the ability of calpain to degrade NFPs could account for the delayed degradation of NFPs in distal segments of severed M-axons maintained in vivo. © 1995 John Wiley & Sons, Inc.     

Modification of shear modulus and creep compliance of fibrin clots by fibronectin

Shear moduli and creep compliances have been measured for four types of clots of human fibrin (about 7 $\text{mg}\text{ml}$) clotted with and without human plasma fibronectin (usually 1.2 $\text{mg}\text{ml}$). Fine clots (with little lateral aggregation of the fibrin protofibrils) were formed at pH 8.5, ionic strength 0.45 ; coarse clots (with substantial lateral aggregation) were formed at pH 7.5, ionic strength 0.15; in both cases with and without ligation by fibrinougase. In fine clots, the addition of fibronectin without ligation scarcely affected the shear modulus; with ligation, the modulus was decreased by a factor of 0.48. In coarse clots, the shear modulus was increased by addition of fibronectin. The increase was by a factor of 2.0 without ligation and by a factor of 2.4 with ligation. Creep and creep recovery in clots formed with and without fibronectin were similar except for the scale factor represented by the change in modulus.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Properties of Wheat Mitochondria. Study of Substrates, Cofactors and Inhibitors

Isolated mitochondria of wheat shoots oxidize α- ketoglutarate, DL-malate succinate and NADH with good relative respiration control and ADP: O ratio. They have high affinity for α-ketoglutarate and NADH as substrates and utilize malate and succinate with a respiration ratio of about one-half of α-ketoglutarate. The average ADP : O ratios approach the expected theoretical values, i.e., 3.6 ± 0.2 for α-ketoglutarate, 1.8 ± 0.2 for succinate, and 2.8 ± 0.2 for malate. The ADP: O ratio with NADH is 1.8 ± 0.2. The maximum coupling of oxidation and phosphorylation is obtained at concentrations of 10 mM, 2 mM, 10 mM and 8 mM for α-ketoglutarate, NADH, malate and succinate, respectively. — Wheat mitochondria have little or no dependence on added cofactors. Mitochondria prepared by our procedure apparently retain sufficient amounts of endogenous cofactors required for NAD-linked systems. FAD⁺ is found to improve succinate oxidation. Cytochrome c does not have any significant effect on respiratory parameters of wheat mitochondria. — Wheat mitochondria are some -what resistant to DNP at 1.7 × 10^-5M. Malonate seems to improve coupling of α-ketoglutarate oxidation. Other Krebs cycle intermediates have been tested on three major substrates of TCA cycle, i.e., α-ketoglutarate, malate and succinate.     

Bivalent binding to gammaA/gamma'-fibrin engages both exosites of thrombin and protects it from inhibition by the antithrombin-heparin complex

Thrombin exosite 1 binds the predominant gamma(A)/gamma(A)-fibrin form with low affinity. A subpopulation of fibrin molecules, gamma(A)/gamma'-fibrin, has an extended COOH terminus gamma'-chain that binds exosite 2 of thrombin. Bivalent binding to gamma(A)/gamma'-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from gamma(A)/gamma'-fibrin clots than from gamma(A)/gamma(A)-fibrin clots. After 24 h of washing, however, both gamma(A)/gamma'- and gamma(A)/gamma(A)-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in gamma(A)/gamma'-fibrin clots than they were in gamma(A)/gamma(A)-fibrin clots. This difference reflects bivalent binding of thrombin to gamma(A)/gamma'-fibrin because (a) it is abolished by addition of a gamma'-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the gamma'-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with gamma(A)/gamma'-fibrin than with gamma(A)/gamma(A)-fibrin clots. Thus, bivalent binding of thrombin to gamma(A)/gamma'-fibrin protects thrombin from inhibition, raising the possibility that gamma(A)/gamma'-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.     

Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β-tubulin

To obtain information on plant microtubule stability to low temperature and Ca²⁺, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβ_s-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβ_s-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβ_s-microtubules. Ca²⁺ enhanced the cold-induced depolymerization of both αβ- and αβ_s-microtubules. However, αβ_s-microtubules were significantly more stable to depolymerization by cold and Ca²⁺ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca²⁺. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.     

Analysis of leaf-rolling behavior ofCaloptilia serotinella (Lepidoptera: Gracillariidae)

The caterpillarCaloptilia serotinella generates the force required to roll leaves by stretching the silk strands it fixes between opposable plant surfaces. The Young's modulus of strands drawn by caterpillars at an average rate of 16 mm s^–1 was 1.1×10⁸ N m^–2. Single strands stretched in a tensiometer had a final Young's modulus of 1.4×10⁹ N m^–2 and withstood a maximum force of 60 × 10^–5 N (i.e., a 60-mg force) before breaking at 30% extension. Strands stretched approximately 14% beyond their equilibrium length by rolling caterpillars exerted an average axially retractive force of 3.2×10^–5 N and drew the leaf 7×10^–3 mm into the roll. During episodes of rolling, the caterpillars spun hundreds of strands capable of generating a collective force in excess of 0.1 N. Potential forces associated with wet contraction of strands were not harnessed by the caterpillar when rolling but subsequent supercontraction of the strands caused them to bind the roll tightly. Caterpillars appeared to facilitate leaf rolling by weakening the midrib with their mandibles.     

Concentration of protein in fibrin fibers and fibrinogen polymers determined by refractive index matching

We have used refractive index matching to determine the concentration of protein in the fibers in fibrin clots and of needlelike crystals of native fibrinogen. Our results are in agreement with those of Carr and Hermans [(1978) Macromolecules 11 , 46–50], as determined by light scattering—namely, that protein makes up about 20% of the volume of the fiber. However, we have found that the protein concentration is strongly dependent on ionic strength. An increase in ionic strength caused a substantial drop in the protein concentration. In a buffer containing 100 mM NaCl, the protein concentration was 26.6–29.8 g of protein per 100 cm³ of polymer, and at 200 mM NaCl it was reduced to 22.1–23.1 g/100 cm³.     

Inhibitory effect of α-hydrazinoornithine on egg cleavage in sea urchin eggs

In fertilized sea urchin eggs which are kept in sea water containing α-hydrazinoornithine (αHO) at a concentration above 1 mM from the time of fertilization, cleavage is delayed markedly. The third cleavage is almost completely blocked by 3 mM αHO. Hydrazine, as well as ornithine, exerts no harmful effect on egg cleavage. αHO causes competitive inhibition of ornithine decarboxylase (ODC) in the egg homogenate. Polyamine levels decrease in fertilized eggs treated with αHO. The addition of ornithine (above 3 mM) to an egg culture containing αHO prevents the αHO-induced delay of cleavage. Putrescine (0.2–0.5 mM), which is the product of the reaction catalyzed by ODC, also relieves egg cleavage from the inhibited state. The same effect occurs in the presence of spermidine (0.2–0.5 mM) or spermine (0.1–0.8 mM). Especially, spermine (0.5 mM) completely cancels the inhibitory effect of αHO on egg cleavage. Egg cleavage is delayed only slightly in the presence of each polyamine (above 2 mM).     

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

Variable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C₃ gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C₄ grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl₂, 1 mM KH₂PO₄, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO₂ fixation by the C₄ mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C₃ mesophyll protoplasts supported CO₂ fixation without added substrates. Rates of CO₂ fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C₄ mesophyll protoplasts were stable when stored at 25°C.     

Electrical properties and excitation-contraction coupling in skeletal muscle treated with ethylene glycol

The contractility of the frog sartorius muscle was suppressed after treatment with a Ringer solution added with ethylene glycol (EGR). No contraction was elicited by nerve stimulation when the muscle was brought back to normal Ringer solution after having been soaked in 876 mM EGR for 4 hr or in 1095 mM EGR for 2 hr. However, the action potential of normal amplitude was generated and followed by a depolarizing afterpotential. The resting membrane potential was slightly decreased from the mean normal value of –91.1 mv to –78.8 mv when 1095 mM EGR was used, and to –82.3 mv when 876 mM EGR was used, but remained almost constant for as long as 2 hr. The afterpotential that follows a train of impulses and a slow change in membrane potential produced by a step hyperpolarizing current (so-called "creep") were suppressed after treatment with ethylene glycol. The specific membrane capacity decreased to about 50% of the control values while the specific membrane resistance increased to about twice the control values Therefore, the membrane time constant remained essentially unchanged. The water content of the muscle decreased by about 30% during a 2 hr immersion in 1095 mM EGR, and increased by about 30% beyond the original control level after bringing the muscle back to normal Ringer. The intracellular potassium content did not change significantly during these procedures. Some differences between the present results and those obtained with glycerol are discussed.