Hydrolysis of erythrocyte membrane phospholipids by a preparation of phospholipase C from Clostridium Welchii. Deactivation of (Ca-2+, Mg-2+)-ATPase and its reactivation by added lipids.

1. Haemoglobin-free erythrocyte ghosts were prepared in 40 imosM bicarbonate buffer, pH 7.4, containing 1 mM EDTA (40 imosM/l mM EDTA). The ghost preparation was highly permeable on preparation but partially resealed on incubation in media containing Ca-2+. 2. A partially purified preparation of phospholipase C from Clostridum welchii caused an increase in observed Mg-2+-ATPase activity, reflecting a change in the permeability of the ghost to substrate. The phospholipase did not decrease Mg-2+-ATPase even at the highest levels tested. Mg-2+-ATPase activity could therefore be used as a permeability indicatior in these experiments. 3. Both (Ca-2+, Mg-2+)-ATPase activities of the ghosts were progressively lost as a result of the phospholipid hydrolysis induced by phospholipase C. 4. When a haemolysin in the commercial preparation was destroyed by heat-treatment, deactivation of the (Ca-2+, Mg-2+)-ATPase and (Na+, K+, Mg-2+)-ATPases were still observed but permeability changes were greatly reduced. 5. The products of phospholipase action were not inhibitory to the Ca-2+, Mg-2+)-ATPase. 6. Lysolecithin brought about a reactivation of the (Ca-2+, Mg-2+)-ATPase which was superimposed upon permeability changes in the preparation. 7. Reactivation of the (Ca-2+, Mg-2+)-ATPase was brought about by a nonlytic, mixed lipid preparation without significant effect upon permeability. 8. Human erythrocyte (Ca-2+, Mg-2+)-ATPase therefore appears to be an enzyme which responds to perturbation of the lipid environment in the membrane and is a "lipid-dependant" enzyme.     

Ecto-adenosine triphosphatase activity at the cholinergic nerve endings of the Torpedo electric organ

Synaptosomes isolated from the electric organ of Torpedo marmorata contain activity of an ATPase which is located at the extracellular face of the plasma membrane. Ecto-ATPase activity can be stimulated independently and to a similar extent by either Ca-2+ or Mg-2+. Apparent Km-values for ATP are 79 microM and 53 microM for Ca-2+ and Mg-2+ respectively. Apparent Km-values for Ca-2+ and Mg-2+ at 1 mM ATP are 0.71 mM and 0.61 mM respectively. The enzyme is also activated by Mn-2+ and GTP can replace ATP as a substrate. Presence of 5'- nucleotidase activity suggests that adenosine is the final hydrolysis product. Thus hydrolysis of nucleotides released during exocytosis of synaptic vesicle contents and purine salvage must be a major role of this ecto-enzyme. We furthermore suggest that the ecto-ATPase may provide the key to understanding the storage of the high energy compound ATP in cholinergic synaptic vesicles. On depolarization of the nerve terminal and exocytosis, ATP represents the signal for activating the ATPase whereby concentrations of Ca-2+ and Mg-2+ are already saturating. Following depolarization induced Ca-2+ influx, a possible function of the ATPase may be the outward transport of Ca-2+ from the nerve terminal.     

The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase.

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.     

Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

The role of Ca2+ on insulin release has been studied by the use of ionophore A23187. The ionophore complexes divalent cations and permits Ca2+ entry into cells by acting as a carrier in the plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the 3rd day of culture. With Ca2+ in the incubation medium the ionophore induced sustained insulin release even in the absence of glucose. Optimal effects of the ionophore were observed at 3 and 10 mug per ml in the presence of 0.3 to 1.0 mM Ca-2+. Under these conditions the insulin release was greater than that caused by 16.7 mM glucose. A graded response was observed to changes in Ca-2+ concentration from 0.1 to 1.0 mM Ca-2+. Higher Ca-2+ concentrations caused a large amount of insulin to be released promptly, but the release was not sustained. Mg-2+ and Sr-2+ were not found to substitute for Ca-2+. Ba-2+ at 0.3 mM stimulated insulin release even in the absence of ionophore. Cyclic adenosine 3':5'-monophosphate was able to increase ionophore-induced insulin release. The alpha-adrenergic effect of epinephrine to inhibit insulin release was not observed in the presence of Ca-2+ and the ionophore, and a stimulatory effect of epinephrine was seen. This unusual stimulatory effect of epinephrine was blocked by propranolol indicating a beta-adrenergic mechanism for epinephrine. It is concluded that Ca-2+, which plays an essential role in the stimulus-secretion coupling, can alone initiate and cause sustained insulin release.     

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyteghosts.

Incubation of etythrocyte ghosts with carbonylcyanide m-chlorophenyl-hydrazone (CCCP) plus Ca-2+ resulted in inactivation of the Ca-2+ -stimulated ATPase activity. Omission of Ca-2+ or lowering of the temperature below 25 degrees C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of beta-mercaptoethanol did not influence the Ca-2+ -dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca-2+ -containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca-2+ -stimulated, Mg-2+ -ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg-2+ -ATPase slightly. CCCP was much more specific for the Ca-2+ -stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca-2+ for inactivation was also quite specific.     

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.     

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.     

Molecular and biological studies on cardiac muscle calcium-binding protein (TN-C).

TN-C was purified from bovine cardiac muscle. In the absence of Ca-2+, cardiac TN-C has an intrinsic sedimentation coefficient of 1.93 S and a molecular weight of 18 000 daltons. Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg-2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyoson. Circular dichroism (CD) studies indicate that cardiac TN-C undergoes a major conformational change upon binding Ca-2+. A similar response is elicited by Sr-2+, whereas Mg-2+ has a much less pronounced effect. The presence of Mg-2+ does not alter the net effects of either Ca-2+ or Sr-2+. Cardiac TN-C is rich in acidic amino acid residues. UV absorption, near UV CD, and fluorimetric studies show that the protein lacks tryptophan and has a relatively high phenylalanine to tyrosine ratio. The results of this study invite direct comparisons with results reported for the skeletal muscle analogue of cardiac TN-C.     

Factors modulating filament formation by bovine glial fibrillary acidic protein, the intermediate filament component of astroglial cells

Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.     

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.     

Assembly properties of tubulin after carboxyl group modification

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.     

Purification of microtubule protein from beef brain and comparison of the assembly requirements for neuronal microtubules isolated from beef and hog.

The polymerization of microtubule protein from beef brain is inefficient under the same conditions which are optimal for the assembly of microtubules isolated from hog brain (0.1 m piperazine-N,N′-bis(2-ethanesulfonic acid) buffer at pH 6.94). In examining the conditions required for microtubule polymerization in both beef brain extract and purified microtuble protein, it was determined that the pH optimum was pH 6.62 or 0.3 pH unit lower than the reported optimum for hog. Other assembly requirements (ionic strength, Mg²⁺ and nucleotide concentration, temperature) remained essentially the same as for hog. By separating and recombining fractions of tubulin and nontubulin components prepared from beef and hog microtubule protein, the requirement for the reduction in pH was found to be due to the tubulin and not to the microtubule-associated proteins. It was also determined that the efficiency of beef tubulin assembly, as measured by the yield of microtubule polymer, decreased rapidly after slaughter with a half-time of 19 min. Furthermore, when the overall efficiency of polymerization was reduced, the extent of assembly at each cycle of purification by disassembly and assembly was also observed to be depressed. The variations in the requirements for neuronal tubulin assembly in two closely related mammals suggest that the conditions required for assembly of microtubule protein in other tissues and cell types may also be different.     

Cation selective promotion of tubulin polymerization by alkali metal chlorides.

A role for charge-based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher-order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+ > K+ > Li+ > Cs+, with optimal concentrations for Na+ at approximately 160 mM. Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of approximately 2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from approximately 0.9 to 0.99, and reverse or prevent urea-induced depolymerization of microtubules. In the presence of microtubule-associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selective persists. Because the selectivity sequence is similar to that of the coil/helix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate-rich C termini of the tubulin monomers.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Tubulin-zinc interactions: binding and polymerization studies

The binding of Zn2+ to tubulin and the ability of this cation to promote the polymorphic assembly of the protein were examined. Equilibrium binding showed the existence of more than 60 potential Zn2+ binding sites on the dimer, including a number of high-affinity sites. The number of high-affinity sites, estimated by using a standard amount of phosphocellulose to remove more weakly bound Zn2+, reached a maximum of 6-7.5 with increasing levels of Zn2+ in the incubation solution. The number also increased with time of incubation at a single Zn2+ concentration. It is suggested that tubulin is slowly denatured in the presence of Zn2+, exposing more binding sites. Cu+ and Cd2+ were effective inhibitors of Zn2+ binding; Mg2+, Mn2+, and Co2+ were much less effective, and Ca2+ was without effect. Zn2+ does not replace the tightly bound Mg2+. GTP reduces the amount of Zn2+ binding under equilibrium conditions and the amount bound to high-affinity sites. Zinc-induced protofilament sheets are produced at a Zn2+/tubulin ratio of 5 in the presence of 0.5 mM GTP, conditions where about two to three Zn2+ ions would be bound to the dimer. At higher GTP concentrations, less assembly occurred, and the products were narrower sheets and microtubules. Zn2+-tubulin, isolated from phosphocellulose, will not assemble unless Mg2+ and dimethyl sulfoxide (Me2SO) or more Zn2+ is added. Broad protofilament sheets, formed from Zn2+-tubulin in the presence of Mg2+ and Me2SO, contain slightly more than one Zn2+ per dimer. It is concluded that Zn2+ stimulates tubulin assembly by binding directly to the protein, not via a ZnGTP complex.     

The red cell membrane contains three different adenosine triphophatases.

Phosphorylated intermediates in the hydrolysis of ATP by human red cell membrane adenosine triphosphatases have been detected using [gamma-32-P]ATP. Intermediates formed in the presence of Mg2+ alone (MG-2+-ATPase), Mg-2+ and Na+ ((Na+,K+)-ATPase), and Mg-2+ and Ca-2+ (Ca-2+-ATPase) were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate at pH 2.4, indicating that these three ATPases are different molecular species. There are roughly 100, 150, and 400 copies per cell, respectively, of the three ATPases.     

Role of tubulin-associated proteins in microtubule nucleation and elongation

Previous experiments have shown that a fraction of microtubule-associated proteins is essential for the self-assembly of microtubules in vitro. When tubulin was titrated with increasing concentrations of these non-tubulin accessory factors, both the rate and extent of polymerization increased in a sigmoidal as opposed to a stoichiometric fashion. The non-tubulin proteins promoted the nucleation of microtubules as determined from the analysis of the kinetics of tubulin selfassembly and the examination of the microtubule length distribution following polymerization. The effect of the non-tubulin factors on microtubule elongation was determined by kinetic experiments in which purified tubulin subunits were added to microtubule seeds and the initial rate of polymerization was measured under conditions where spontaneous self-assembly was below detectable levels. In addition, microtubule growth was also observed when isolated flagellar axonemes were incubated with purified tubulin subunits indicating that the non-tubulin factors were not an absolute requirement for elongation. Analysis of the data in terms of the condensation mechanism of microtubule assembly indicated that the non-tubulin proteins stimulated the growth of microtubules not by increasing the rate of polymerization but by decreasing the rate of depolyerization. The mechanism by which these accessory factors promote tubulin assembly may be summarized as follows: under the conditions employed, they are required for tubulin initiation but not for elongation; the factors affect the extent and net rate at which polymer is formed by binding to the polymer, thereby stabilizing the formed microtubules and consequently shifting the equilibrium to favor assembly.     

Further studies on the formation of cardiolipin and phosphatidylglycerol in rat liver mitochondria. Effect of divalent cations and the fatty acid composition of CDP-diglyceride.

The divalent cation requirement for mitochondrial cardiolipin biosynthesis has been further investigated. The relative order of divalent cation activity was Co-2+ greater than Mn-2+ greater than Mg-2+. Cardiolipin was not formed in the incubations with Zn-2+, Fe-2+, Cu-2+, Hg-2+, and Ca-2+. Cardiolipin synthesis in the presence of optimal cincentration of Co-2+ was inhibited by Ca-2+. A series of CDP-diglycerides was synthesized having differences in fatty acid chain lenth and degree of unsaturation. These compounds were tested in mitochondrial cardiolipin and phosphatidylglycerol synthesis. Although there were some minor differences between phosphatidylglycerol and cardiolipin synthesis, in general, saturated shorter chain CDP-diglycerides (dilauroyl and dimyristoyl) were better substrates than the longer chain dipalmitoyl and distearoyl homologues. Introduction of double bonds into distearoyl CDP-diglyceride resulted in more rapid rates of synthesis (e.g. dioleoyl and dilinoleoyl CDP-diglyceride). Significance of the results is dicussed with regard to possible mechanisms of linoleic acid incorporation into rat liver cardiolipin.     

K+ and Mg-2+ net fluxes in relation to zero [Ca-2+] perfusion and subsequent cardiac contracture.

Following within 45 sec after the development of contracture induced by restoring normal ionic composition perfusion conditions after a 12 min period of mechanical arrest in the rabbit heart caused by zero [Ca-2+] perfusion, there is an explosive efflux of K+ and Mg-2+. After shorter periods of Ca-2+-lack arrest, the restoration of [Ca-2+] to normal causes recovery of rhythmic contraction and no K+ efflux. The K+ and Mg-2+ effuxes are ascribed to the effects of the contracture itself and not simply to the loss of Ca-2+ during zero [Ca-2+] arrest nor to the restoration of normal perfusate [Ca-2+], except insofar as the latter operates to induce the contracture. It is suggested that cell membrane permeability progressively increases during zero [Ca-2+] arrest and that an abnormally large influx of Ca-2+ after restoration of normal perfusate [Ca-2+] induces the contracture.