Vincent du Vigneaud

l-Penicillamine and rat liver transaminase activity

Abstract Addition of l -penicillamine to liver transaminase systems in vitro causes an inhibition of enzyme activity. The inhibitory effect can be reversed by the addition of increasing amounts of pyridoxal 5-phosphate. Data are presented to indicate a chemical combination between l -penicillamine and pyridoxal 5-phosphate at pH 7.4. The inhibitory effect of l -penicillamine in vitro and in vivo is dependent upon the presence of both a free sulfhydryl and amino group in the penicillamine molecule.

An increased vitamin B6 requirement in the rat on a diet containing l-penicillamine

Abstract Addition of l -penicillamine to the diet of the white rat has been found to increase the requirement for vitamin B6. The presence of l -penicillamine in the diet results in a gross symptomatology similar to that reported for B6-deficient animals. Growth inhibition due to l -penicillamine can be reversed by the administration of higher levels of pyridoxine either by injection or by incorporation in the diet. l -Penicillamine-treated rats excrete large amounts of xanthurenic acid after a test dose of tryptophan, and the excretion is suppressed when additional pyridoxine is administered simultaneously with l -penicillamine. The possible metabolic relationship between l -penicillamine and pyridoxine is discussed.

An anti-vitamin B6 action of L-penicillamine.

Abstract The effect of l -penicillamine administration on rat liver transaminase activities and on the excretion of vitamin B 6 activity in the urine was investigated. l -Penicillamine caused a reduced activity of the alanine-glutamic and aspartic-glutamic transaminases. Reversal of the inhibition was accomplished by the simultaneous administration in vivo of pyridoxine or by addition in vitro of pyridoxal 5-phosphate. Inclusion of l -penicillamine in the diet resulted in a marked increased excretion of vitamin B 6 activity in the urine. l -Penicillamine apparently causes a rapid loss of vitamin B 6 from the body which strongly suggests an anti-vitamin B 6 role in vivo for l -penicillamine.

On the Nature of Oxytocin and Vasopressin from Human Pituitary.

Summary The oxytocic and pressor activities of the human posterior pituitary gland were separated chromatographically after preliminary purification. The behavior of purified components on ion exchange chromatography, paper chromatography and paper electrophoresis indicated that they possessed the properties of oxytocin and arginine vasopressin. The amino acid composition of the oxytocin and vasopressin isolated from the human glands was determined. It is evident that the oxytocin and vasopressin found in the human pituitary are identified as oxytocin and arginine vasopressin.

Oxytocin and milk ejection.

Abstract 1. 1. Oxytocin isolated from the posterior pituitary gland free of any detectable amount of vasopressin and by available physical and chemical criteria essentially a pure chemical compound has milk-ejection activity in recently parturient women who have milk in their breasts. 2. 2. A synthetic octapeptide amide, in so far as has been tested, indistinguishable from naturally occurring oxytocin in other properties, has milk-ejection activity in similar puerperal women indistinguishable from the naturally occurring oxytocin, thus affording conclusive proof that oxytocin has both oxytocic and milk-ejection activities inherent in its molecular structure. 3. 3. Milk ejection occurs consistently in these women when approximately 0.5 unit of oxytocin is administered intravenously, or when 2 units of oxytocin are administered intramuscularly.

The synthesis of the histidine analog of the vasopressins

Abstract An analog of the vasopressins has been prepared containing histidine in place of the lysine or arginine occurring in the natural hormone. The synthesis of this cyclic octapeptide amide involved the preparation of the protected nonapeptide, S -benzyl- N -tosyl- l -cysteinyl- l -tyrosyl- l -phenylalanyl- l -glutaminyl- l -asparaginyl- S - benzyl- l -cysteinyl- l -prolyl- l -histidylglycinamide, cleavage of the protecting groups with sodium in liquid ammonia, and oxidation of the resulting sulfhydryl nonapeptide to the cyclic octapeptide amide. The histidine vasopressin possessed only 1.5 units of pressor activity/mg. as compared with approximately 300 and 400 units in the natural hormones. It would thus appear that the relative basicity of the amino acid residue in the penultimate position in the side chain of the posterior pituitary hormones has a considerable influence on the degree of pressor activity of these compounds.

EXPERIENCES IN THE POLYPEPTIDE FIELD: INSULIN TO OXYTOCIN

It has been my privilege for the past 35 years to have witnessed, anL a t times participated in, a truly exciting development in the field of biochemistry. The development I have in mind involved: first, the recognition and acceptance that a hormone could be a protein or polypeptide; second, the working out of the organic chemical structures of certain polypeptide and protein hormones; third, the demonstration by synthesis of the authenticity of the structures postulated for certain lower molecular weight polypeptide hormones; and, finally, the present stage where the structures of these polypeptides can be modified almost a t will by synthetic means in an effort to understand the specificity of the structures involved-by the replacement of constituent amino acids by other amino acids to produce various analogues and derivatives. Parallel to this development of our knowledge of the structure of the polypeptide hormones has been a comparable development, the recognition that enzymes are proteins, culminating in the recent achievement of the establishment of the structure of one of these, ribonuclease. Closely following the unfolding of our knowledge of hormones and enzymes have been the exciting developments in the field of viruses. We are even beginning to get a glimpse of the interrelationship between a protein and a nucleic acid in their role in virus activity. Before long we shall know the structures of certain virus proteins. Already that is well on the way. Synthesis may well follow. These are truly exciting times. It is appropriate that we consider in this symposium certain phases of this rapidly expanding polypeptide field. Perhaps there is some advantage in pausing initially to reflect on where we have been, to ponder on where we are, and to speculate on where we are going. Karl Folkers has asked me to do this and has particularly urged me to discuss from personal experience the development of the protein and polypeptide hormone field as I have lived through it. As one surveys the protein and polypeptide hormone field today, it is almost unbelievable how hard it was some 35 years ago for the workers in the field to accept the idea that a protein could be a hormone or, to put it the other way around, that a hormone could be a protein. Today we have arrived a t the point where we have several polypeptide hormones already synthesized, and we know the structures of several other polypeptide and protein hormones and even one enzyme. There is little doubt that in the not too far distant future the protein hormones and enzymes whose structures are established by degradative studies will succumb to the synthetic attack of the peptide chemist. I t is, of course, a tradition of organic chemistry that before final acceptance of any structure, synthesis by unambiguous procedures must be accomplished. I t is reassuring to realize that the organic chemistry of peptides is now reaching the stage where synthetic proof of structure can be called upon to check the

4-Leucine-Oxytocin: Natriuretic, Diuretic, and Antivasopressin Polypeptide

During water diuresis in anesthetized rats, 4-leucine-oxytocin increased the urine output and the rates of sodium and chloride excretion. The potassium excretion rate was only slightly increased. During vasopressin-suppressed water diuresis, 4-leucine-oxytocin produced similar effects on urine and electrolyte excretions. In addition, it inhibited the vasopressin-induced free-water reabsorption, and it could reverse reabsorption to freewater clearance.

Synthesis and Some Pharmacological Properties of a Peptide Derivative of Oxytocin Glycyloxytocin.

Summary 1) The protected decapeptide carbobenzoxyglycyl - S - benzyl - l - cys-teinyl - l - tyrosyl - l - isoleucyl - l - gluta-minyl - l - asparaginyl - S - benzyl - l - cysteinyl - l - prolyl - l - leucylglycinamide was synthesized by coupling of the p-nitrophenyl ester of carbobenzoxyglycine with S-benzyl-l - cysteinyl - l - tyrosyl - l - isoleucyl - l -glutaminyl - l - asparaginyl - S - benzyl - l -cysteinyl - l - prolyl - l - leucylglycinamide. Removal of the protecting groups followed by oxidation to the disulfide gave glycyloxytocin which was isolated by countercurrent distribution. 2) The pharmacological effects of this peptide derivative of oxytocin were investigated by testing it for avian depressor, pressor, and rat uterine-contracting activities. The sample of glycyloxytocin possessed only a slight degree of these 3 activities. It was found to exert a marked inhibitory effect on the avian depressor activity of oxytocin. On the other hand, the glycyloxytocin did not show inhibitory action against oxytocin on the isolated rat uterus or against oxytocin or vasopressin in the assay for pressor activity in the rat. 3) Thus, the attachment of a glycyl residue to the free amino group of oxytocin causes almost complete loss of its avian depressor, rat uterine-contracting, and pressor effects. In addition, it is of considerable interest that this peptide derivative of oxytocin exerts a marked inhibitory action upon the avian depressor effect of the hormone.

Enzymatic Evidence for Intrinsic Oxytocic Activity of the Pressor-Antidiuretic Hormone

Summary 1. It has been shown that trypsin had no effect on the activity of oxytocin but destroyed both the pressor and oxytocic activities of arginine- vasopressin. These conclusions offer enzymatic evidence, in addition to the evidence already obtained from analytical and electrophoretic studies, for the inherent oxytocic activity of arginine-vasopressin. 2. It has also been demonstrated that a lysine-vasopressin preparation possessed oxytocic activity and that this activity was destroyed with the pressor activity by trypsin.