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Compound 1,4-(4-nitrophenoxy)-2-keto-n-butylphosphoric acid, was synthesized and shown to undergo elimination ofp-nitrophenol in aqueous solution at pH 7.0 to form 2-keto-3-butenylphosphoric acid ( 2 ) at a rate 50 times greater than hydration to 2-keto-4-hydroxybutylphosphoric acid (4) occurs. A term in the rate expression for 12-4 2 is ascribed to an intramolecular proton abstraction of the 01 proton by the phosphate oxygen. Rabbit muscle aldolase does not catalyze the production of even 1 equiv of p-nitrophenol in the presence of excess 1. The elimination product, 2, however, causes time-dependent inhibition of the enzyme. The pH and concentration dependence of this reaction was studied, as well as protection from inhibition by 2 using dihydroxyacetone phosphate. Formation of the mixed disulfide of 6-mercaptopropionate with an active-site thiol group also affords protection from inhibition by 2. The rate of hydrolysis of the enzyme-inhibitor complex was measured by two methods, and this demonstrated that the equilibrium constant for binding of 2 was IO9 M-' at pH 7. Compounds 1 and 4 were shown to bind to aldolase but not undergo reaction. A mechanism for inactivation is proposed in which 2 forms an iminium ion with the lysine function, followed by a Michael-type addition of a thiol to afford the inactive complex. IntroductionWe have previous:;. shown' that 2-keto-3-but en ylphospha te (2) inhibits rabbit muscle aldolase in an active site directed, time-dependent manner. A logical explanation of this behavior2 is that 2 condenses with the €-amino group of lysine present at the active site,3 followed by attack of an active-site nucleophile, as shown in Scheme I. The formation of the iminium ion would be analogous to the condensation of the natural substrate dihydroxyacetone phosphate (DHAP) with the enzyme. Aldolase does not catalyze the steady-state production of p-nitrophenol from 1, even though a reasonable mechanism may be written using precedentedIn this paper we describe the details of the syntheses and reactivity of 1,2, and 4 and their interactions with the enzyme. In particular, we wished to determine if a single turnover of 1 to 2 was catalyzed by aldolase and also to determine if the inhibition by 2 was reversible. It was also desirable to see if an active site directed thiol reagent would prevent inhibition in order to determine if a cysteine could be implicated as the reacting nucleophile.A study of both the forward and reverse rates of reaction might provide some generally useful information about the free energy of interaction covalent inhibitors with enzymes. Many of the suicide type of inhibitors or those that react further after a Michael-type reaction would not be amenable to study in

The superficial flexor muscle of the crayfish is a neuromuscular system in which the neurons form position-dependent connectivity patterns with the muscle fibers. This system could be formed with the help of a single medial-to-lateral gradient during development that embodies positional information. To test this gradient hypothesis we changed the nerve's normal medial entry point into the muscle by transplanting it to the middle of the muscle sheet. When all the muscle fibers were present in the target area, most of the neurons studied passed through a stage during regeneration in which they showed preference for either medial or lateral synapse formation. Those neurons that in normal animals innervated preferentially the medial fibers showed a medial preference for new contacts; the neuron that normally innervated the lateral fibers showed a lateral preference for new contacts; the neuron that normally innervated everywhere regenerated equally well into both medial and lateral fibers. Therefore, these neurons are able to detect information regarding their position within the muscle mass and respond to it by preferential synapse formation. The effect of a positional gradient could not be detected when half of the target field was removed prior to regeneration. In this instance, the neuron that innervated the missing target area now regenerated to almost all the available fibers. It is suggested that the interplay of positional cues with other factors at different points in time could determine the final connectivity patterns formed by these cells.

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.