Photooxidation of dinitrophenylhistidine-200 human carbonic anhydrase B.

Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis.     

The stimulation of collagenase production in rabbit articular chondrocytes by interleukin-1 is increased by collagens

Osteoarthritis is characterized by a loss of articular cartilage due at least in part to the action of degradative enzymes secreted by chondrocytes. We have investigated the effect of type II collagen from cartilage and interleukin 1 on collagenase production in cultures of rabbit articular chondrocytes. Interleukin 1 alone stimulated the chondrocytes to secrete collagenase but this response was increased as much as fivefold by the addition of rabbit type II collagen. Bovine type II and chick type I collagens were also stimulatory. The native form of the collagens was not required since denatured collagens and purified chick type II alpha chains were effective. The observed effects of collagens and interleukin 1 may contribute to the progressive nature of osteoarthritis.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia.

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.     

H2 histaminic receptors in rat cerebral cortex. 1. Binding of [3H]histamine

Saturable binding of [3H]histamine in equilibrium with homogenates of rat cerebral cortex reveals Hill coefficients between 0.4 and 1.0, depending upon the conditions. Data from individual experiments are well described assuming one or two classes of sites. Only the sites of higher affinity (KP1 = 3.9 +/- 0.5 nM) are observed when binding is measured by isotopic dilution at a low concentration of the radioligand (less than 1.5 nM) in the presence of magnesium or by varying the concentration of the radioligand. The sites of lower affinity (KP2 = 221 +/- 26 nM) appear during isotopic dilution at higher concentrations of the radioligand or at lower concentrations either upon the addition of guanylyl imidodiphosphate (GMP-PNP) or upon the removal of magnesium. Estimates of the second- and first-order rate constants for association and dissociation of [3H]histamine agree well with KP1. Apparent capacities corresponding to KP1 and KP2 are of the order of 100 ([R1]t) and 1300 pmol/g of protein ([R2]t), respectively. Simple interconversion cannot account for the changes in binding that occur upon adding GMP-PNP or removing magnesium, since the increase in [R2]t exceeds the decrease in [R1]t. Moreover, the apparent amount of high-affinity complex exhibits a biphasic dependence on the concentration of [3H]histamine; an increase at low concentrations is offset by a decrease that occurs at higher concentrations. The latter appears to be positively cooperative and concomitant with formation of the low-affinity complex. These and other observations indicate that the binding of histamine is inconsistent with models commonly invoked to rationalize the binding of agonists to neurohumoral receptors. GMP-PNP and magnesium reciprocally alter capacity at the sites of higher affinity, however, and the reduction caused by GMP-PNP reflects a substantial increase in the rate constant for dissociation at the sites that appear to be lost. The sites labeled by [3H]histamine thus reveal the properties of neurohumoral receptors linked to a nucleotide-specific G/F protein.     

H2 histaminic receptors in rat cerebral cortex. 3. Inhibition of [3H]histamine by H2 agonists

The binding of [3H]histamine to H2 receptors in homogenates of rat cerebral cortex is inhibited by 11 H2 agonists in a characteristic and unique manner. At low concentrations of the radioligand (less than 1.5 nM), the inhibitory profiles of individual agonists (A) are distinctly biphasic; specific binding is well described in most cases by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1. Maximal inhibition is the same for all agonists. Since values of F2 vary from 0.42 to 0.90, the agonist appears to determine the equilibrium distribution of receptors between two states of affinity. Ratios of apparent affinity (K2/K1) vary from 204 to 3 090 000, and there is no correlation between values of K1 and K2. Compounds lacking H2 activity, including structural analogues of histamine and dimaprit, reveal a Hill coefficient of 1 and inhibit the radioligand only weakly. For six agonists, values of K2 agree and correlate well (P = 0.00047) with H2 pharmacological potency (EC50) in the guinea pig right atrium; for the others, K2 is less than EC50 by 15-61-fold. Four observations suggest that the inhibition corresponding to F1 is allosteric and cooperative: the dissociation constant of the radioligand appears to vary in the presence of an unlabeled agonist, absolute levels of binding corresponding to F1, as defined by dimaprit, decrease at higher concentrations of [3H]histamine, F1 for dimaprit is reduced from 0.48 to 0.32 by 2-methylhistamine (F1 = 0.27) at a concentration of 20 nM (approximately K1(0.5) K2(0.5) for 2-methylhistamine), but the increase in K1 for dimprit is at least 100-fold less than expected from competitive effects, and 1 equiv of some agonists appears to preclude access of [3H]histamine to more than 1 equiv of receptors, with no evidence that an appreciable fraction of the unlabeled drug is bound. Noncompetitive effects also may account in part for the inhibition corresponding to F2.     

Inhibitor of protein synthesis blocks longterm behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effect of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.