Joseph V. Auditore

Cardiac (microsomal) Na + K adenosinetriphosphatase and its possible relationship to the active Na + K transport system☆

Abstract An ATPase has been found in the high-speed sediment (52,000 × g ) isolated from a rabbit ventricle sucrose homogenate containing 0.1% deoxycholate, whose activity depends on the relative concentrations of four naturally occurring cations, namely, Na, K, Mg, and Ca. In the presence of Mg, the addition of Na and K separately stimulated enzyme activity slightly. When Na and K are present together with Mg, an intense stimulation of enzyme activity occurs with the optimum Na:K ratio being approximately 10:1. In the presence of isotonic Na + K solutions, the K m values for Na and K are 1 and 56 m M respectively. High K in the presence of Na inhibited ATPase activity, and the inhibition was analyzed as the competitive type. Demonstrated is the complex relationship that exists when Na and K are both present. Ammonium can substitute for K but not for Na. Calcium inhibited the Na-K ATPase. The influence of G-strophanthin in the presence of Mg, Mg + Na, Mg + K, and Mg + Na + K is reported. Half-maximal inhibition occurs at 6.3 × 10 −7 M G-strophanthin. The similarities between the Na-K activated ATPase and the active, linked, Na + K transport system are discussed.

Isolation, purification, and probable structural configuration of N-acetyl aspartyl glutamate in human brain ☆

Abstract An acetylated dipeptide composed of equimolar quantities of aspartic acid and glutamic acid has been isolated in pure form from human brain by a combination of ion exchange and paper chromatographic techniques. Hydrazinolysis and acid hydrolysis studies indicate that the peptide is N -acetyl aspartyl glutamate. Preliminary synthesis studies suggest that the peptide may be of the alpha rather than the beta structural configuration.

Investigation of unidentified pharmacologically active substances in the human central nervous system

An unidentified substance with acetylcholine-like activity has been isolated from human brain by Chromatographie procedures. The substance is more active on the frog reclus preparation than on the guinea pig ileum. It has anRitf value of 0.65 ± 0.05 in BuOH: EtOH:Ac:H2O. An amino acid analysis of material isolated from this region of the chromatogram revealed two major ninhydrin peaks corresponding to leucine (0.112 μmoles) and isoleucine (0.052 μmoles). Two additional prominent ninhydrin peaks appeared in an equivalent amount of an acid hydrolyzate which corresponded to aspartic acid (1.925 μmoles) and glutamic acid (1.709) μmoles). The amounts of leucine and isoleucine were of the same order as those in the unhydrolyzed sample. Only trace amounts of other amino acids were found. Dinitrophenylation experiments did not give rise to either DNP-glutamic acid or DNP-aspartic acid on hydrolysis. Hydrozinolysis indicated glutamic to be a C-terminal amino acid. The hydrolyzed material possessed no pharmacological activity. The active substance was temporarily presumed to be an acetylated dipeptide of aspartic and glutamic acid.

Sodium-potassium activated G-strophanthin sensitive ATPase in cardiac muscle.

Summary An ATPase has been demonstrated in water extracts and microsomal suspensions prepared from rabbit ventricles. It has an absolute requirement for Mg and it can be further stimulated by Na and K. The Na + K active component is susceptible to inhibition by G-Strophanthin. Ageing increases the Mg + Na + K/Mg ATPase ratio.

OCCURRENCE OF N‐ACETYL‐l‐GLUTAMIC ACID IN THE HUMAN BRAIN

A substance apparently identical with N‐acetyl‐l‐glutamic acid was isolated from an aqueous extract from human brain by a combination of paper and ion exchange chromatography. The isolated substance does not react with ninhydrin reagent but yields glutamic acid upon acid hydrolysis. Acetyl hydrazide was identified by paper chromatography of hydrazinolysates of the isolated substance and N‐acetyl‐l‐glutamic acid. The configuration was determined with l‐specific hog kidney acylase.

A NA + K-ACTIVATED MGATPASE IN RABBIT KIDNEY MITOCHONDRIA.

Abstract A concealed Na+K-activated MgATPase has been uncovered in rabbit kidney mitochondria. The Na+K-activated component but not the Mg component is inhibited by G-strophanthin. The enzyme apparently resides in the membrane structure of mitochondrion.

Evidence for the presence of N-Acetyl-α-l-aspartyl-l-glutamate in human brain

Abstract A peptide composed of equimolar quantities of glutamic and aspartic acids was isolated from a protein-free extract of human brain by a combination of ion-exchange and paper chromatography. Hydrazinolysis studies established that glutamic acid was C-terminal and that the amino group of the aspartyl residue was substituted with an acetyl group. To determine which aspartyl carboxyl group was linked to glutamic acid, N -acetyl-α- and N -acetyl-β-aspartyl glutamate were synthesized. When the partial acid hydrolyzates of the synthesized acetylated peptides were chromatographed on the amino acid analyzer, two distinct elution patterns were observed. The elution pattern of the brain dipeptide resembled the alpha rather than the beta derivative. When acetylated amino acids prepared from brain dipeptide were incubated with hog l -specific kidney acylase, aspartic and glutamic acids were liberated.

Localization and characterization of a lipase in rat adipose tissue

A lipolytic enzyme from rat adipose tissue has been studied in a cell-free system. Most of the activity toward triglyceride was recovered in a fraction that sedimented at 105,000 g for 18 hours. Properties of the high-speed sediment included: (a) a strong pH dependency, with optimum activity at pH 7.4; (b) instability, half-life of 2 1/2–3 days; (c) partially inactivated by heat; (d) activated by SH reagents; (e) stimulation by epinephrine was observed only in a preparation which had become partially or totally inactivated during storage; (f) neither the whole homogenate nor the fully active fractions were stimulated by epinephrine.

Isolation of N-acetylglutamine, pyroglutamic acid and the butyl ester of pyroglutamic acid from human brain.

N‐acetyl‐l‐glutamine, pyroglutamic acid, and the butyl ester of pyroglutamic acid were isolated in pure form from an aqueous extract of human brain. These compounds were isolated by combination of paper and ion exchange chromatography. The isolated substance identified as N‐acetyl‐l‐glutamine did not react with the ninhydrin reagent but yielded glutamic acid and ammonia upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography from hydrazinolysates of the isolated substance. The glutamic acid liberated by hydrolysis had the l‐configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N‐acetyl‐l‐glutamine. A large amount of pyroglutamic acid and a substance identical with the butyl ester of pyroglutamic acid were isolated in pure form. The results of our studies suggest that pyroglutamic and the butyl ester derivative were artifacts formed during the isolation and purification procedures.

N-acetyl-L-asparagine in human brain ☆

Abstract A substance identical with N-acetyl-L-asparagine was isolated in pure form from an aqueous extract of human brain. The substance was isolated by a combination of an ion exchange and paper chromatography. The substance reacts avidly with chlorine-starch-iodide but does not react with ninhydrin. Upon acid hydrolysis it yields only aspartic acid and ammonia. An acetyl hydrazide was identified by paper chromatography. The unknown had the L-configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for authentic N-acetyl-L-asparagine.