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Tritium-labeled pyridoxamine, pyridoxal 5′-phosphate, and pyridoxine 5′-phosphate were prepared and administered intravenously to mice, and the distribution of isotope in liver and carcass between pyridoxine, pyridoxine 5′-phosphate, pyridoxal, pyridoxal 5′-phosphate, pyridoxamine and pyridoxamine 5′-phosphate was determined at different times after administration. The vitamin B6 forms were extracted from the tissues by perchloric acid and separated by ion exchange chromatography. The results strongly suggest that phosphorylation is the first step in the conversion of pyridoxamine as well as of pyridoxine to the active coenzyme form of the vitamin. After an initial period of equilibration, pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate accounted for more than 90% of recovered isotope in liver and for 80 to 90% of recovered isotope in carcass. The ratio between these compounds was about 2:1 in both liver and carcass. About 10% of the total isotope in carcass appeared as pyridoxal. The available experimental data obtained after administration of different forms of vitamin B6 were found to fit a kinetic metabolic model which assumes an equilibration between pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate but essentially unidirectional reactions in other metabolic conversions.

A soluble fraction from the isthmus region of hen oviduct catalyzes the liberation of SO42- from UDP-N-acetylgalactosamine-4-sulfate (the nucleotide that accumulates in the same tissue during egg formation). The enzyme is purified some 88-fold and is shown to catalyze the reactions: (a) UDP-N-acetylgalactosamine-4-sulfate → UDP-N-acetylgalactosamine plus SO42-; and (b) UDP-N-acetylgalactosamine-4,6-disulfate → UDP-N-acetylgalactosamine-6-sulfate plus SO42-. N-Acetylgalactosamine-4,6-disulfate is desulfated in a similar way but at approximately 13% of the rate of UDP-N-acetylgalactosamine-4-sulfate hydrolysis. The enzyme does not act upon p-nitrophenyl sulfate, N-acetylgalactosamine-4- and 6-sulfate, glucose 6-sulfate, galactose 3- and 6-sulfate, glucan sulfate, chondroitin sulfates, dermatan sulfate, keratan sulfate, heparan sulfate, and unsaturated disaccharide mono- and disulfates derived from chondroitin sulfates. There are striking differences in the sulfatase activity among the anatomically distinct regions of the oviduct: the specific activity of the isthmus extract being 7 to 12 times greater than the values of the magnum and uterus extracts. A somewhat similar activity is detected in the extracts of rat liver and kidney, but available evidence suggests that the activity in these extracts is due to the action of an arylsulfatase which is different from the isthmus sulfatase in catalyzing the cleavage of p-nitrophenyl sulfate. We previously described the occurrence in the isthmus of specific sulfotransferases which catalyze UDP-N-acetylgalactosamine-4-sulfate and UDP-N-acetylgalactosamine-4,6-disulfate formation, respectively. The present finding of a sulfatase which acts preferentially on these sulfated sugar nucleotides gives additional support to the idea that these nucleotides have an important biological role.

Two fractions of adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase (protein kinase B1 and B2) and one fraction of cyclic AMP-independent protein kinase (protein kinase B0) are resolved and partially purified from rat liver cytosol treated with diisopropyl fluorophosphate by ammonium sulfate fractionation followed by DEAE-Sephadex and hydroxylapatite column chromatography. Analysis with gel filtration on a Sephadex column reveals that protein kinase B0 shows a single peak with themolecular weight of approximately 3 x 104, whereas protein kinase B1 is a mixture of two, major and minor, species of cyclic AMP-dependent protein kinases with the molecular weights of 1.8 x 105 and 1.2 x 105, respectively. However, the minor species appears to be an artifact due to proteolysis during the isolation procedure. Protein kinase B2 is also a mixture of multiple cyclic AMP-dependent protein kinases with the molecular weights ranging from 1.0 x 105 up to more than 2.0 x 105. This heterogeneity does not seem to represent simply artifact due to proteolysis during the isolation procedure. Each of the cyclic AMP-dependent protein kinases is shown to be an inactive form of protein kinase, the activity of which is inhibited by association of regulatory protein (R-protein). R-protein per se is catalytically inactive. Cyclic AMP activates such inactive form by binding to the R-protein molecule in an allosteric manner, resulting in the release of active protein kinase. All active protein kinases thus released from both protein kinase B1 and B2 are indistinguishable from protein kinase B0, and show the molecular weight of 3 x 104. The active protein kinases show similar kinetic properties and behave in an identical manner upon chromatography on a hydroxylapatite column. These kinases are not stimulated nor inhibited by the cyclic nucleotide, and phosphorylate the same specific seryl and threonyl residues of histone. An available evidence suggests that the multiple cyclic AMP-dependent protein kinases differ from each other in their associated R-proteins, although the exact identity of the active protein kinases may be explored by further investigations. Protein kinase B1 is shown to be reconstituted from protein kinase B0 and each of the R-proteins with the molecular weights of 1.5 x 105 and 9 x 104, although the latter R-protein is probably an artifact due to proteolysis. These R-proteins are partially purified from rat liver cytosol. However, attempts to reconstitute protein kinase B2 have been unsuccessful. Most of the R-protein is unstable and decomposed to a smaller protein with the molecular weight of about 4 x 104. Protein kinase B1 and B2 show closely similar properties, but their affinities for cyclic AMP are slightly different.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

We investigated how changes in tryptophan pyrrolase activity and tryptophan loads affect the breakdown of tryptophan was estimated by injecting rats with [ring-2-14-C]tryptophan and measuring respiratory 14-CO2. We concluded, contrary to previous reports, that induction of tryptophan pyrrolase definitely will increase the rate of tryptophan breakdown. Tryptophan loads also increase tryptophan breakdown even in circumstances where there is no increase in tryptophan pyrrolase activity, presumably by increasing the saturation of the enzyme. After a tryptophan load (50 mg per kg) the increase in liver tryptophan concentration lasts only 30 min. The rapid return of liver tryptophan to normal may be due partly to the high turnover rate of liver tryptophan. We estimate that tryptophan pyrrolase degrades tryptophan in vivo at a rate that is equivalent to the whole liver tryptophan concentration in 7.5 min or less.

Rat liver microsomes are capable of catalyzing the acylation of 1-palmitoyl-sn-glycero-3-phosphoryl-rac-glycerol for phosphatidylglycerol formation. The requirements for the reaction appear to be the same as the analogous reaction for lecithin synthesis. In contrast, however, to oleoyl-CoA:1-palmitoyl-sn-glycero-3-phosphorylcholine acyltransferase, which has a pH optimum of 7.0, oleoyl-CoA:1-palmitoyl-sn-glycero-3-phosphoryl-rac-glycerol acyltransferase functions optimally at pH 9.0. The latter is also distinguishable by being more stable at the alkaline pH than the former. The data indicate that in addition to positional specificity and acyl group preferability, acyl-CoA:acyl glycerophospholipid acyltransferases also possess phosphorylalcohol group specificity.

Diphtheria toxin catalyzes the transfer of the adenosine diphosphate ribose portion of nicotinamide adenine dinucleotide to aminoacyl transferase II purified to a homogeneous state from rat liver. A stoichiometric amount of nicotinamide is released simultaneously. The ADP-ribosylation of aminoacyl transferase II results in a concomitant inactivation of this particular enzyme, which has a function in the translocation of peptidyl transfer RNA on ribosomes. The reaction is reversible, since toxin catalyzes the formation of NAD from ADP-ribosylated aminoacyl transferase II and nicotinamide with a concurrent restoration of the enzyme activity. The equilibrium constant of the ADP-ribosylation reaction is determined to be 6.3 x 10-4. From this constant the free energy of hydrolysis of the ADP-ribose transferase II linkage is calculated to be approximately 4000 cal per mole at pH 7 and 25°. The ADP-ribosylation and reverse reactions are most active at pH 8.5 and 5.2, respectively. The apparent Michaelis constants for NAD and nicotinamide are 5 x 10-6 m at pH 7.4 and 5 x 10-4 m at pH 5.2, respectively. No other derivatives of NAD so far tested are capable of replacing NAD except thionicotinamide adenine dinucleotide. Diphtheria antitoxin inhibits the reaction, and toxoid is inactive. So far aminoacyl transferase II is the sole protein species found to be ADP-ribosylated in the supernatant fraction from rat liver. Toxin catalyzes neither hydrolysis of NAD nor the exchange reaction between NAD and nicotinamide-14C.