Rat brain extract was found to catalyse the formation of 5-hydroxyindoleacetic acid from L-5-hydroxytryptophan in the presence of α-ketoglutarate, pyridoxal phosphate, NAD⁺ and iproniazide as a monoamine oxidase inhibitor. 5-Hydroxyindoleacetic acid was not formed in the absence of α-ketoglutarate. The omission of NAD+ from the incubation mixture decreased 5-hydroxyindole- acetic acid formation. These findings show that 5-hydroxyindoleacetic acid was produced from L-5-hydroxytryptophan via a transamination pathway which does not contain 5-hydroxytryptamine but 5-hydroxyindolepyruvic acid as an intermediate. The physiological significance of this pathway is discussed.

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Recently, Guillemin and his coworkers (1973) presented a tetradecapeptide structure for somato- tropin-release inhibiting factor (SRIF) and reported that the synthetic replicate corresponding to the proposed sequences possessed identical activities with that of natural purified SRIF. We have perform- ed the synthesis of this peptide and examined its effects on GH release and cyclic AMP formation in rat anterior pituitary gland. The concentration of cyclic AMP in tissue was determined by competi- tive protein binding assay and GH in the medium or in plasma were assayed using rat GH radio- immunoassay kit provided by NIAMDD. Synthetic SRIF (10ng/ml) decreased significantly the basal level of cyclic AMP in rat anterior pituitary incubated in KRB buffer containing glucose (1mg/ml) and theophylline (10^-2M) and showed marginal inhibition of GH release from the pituitary gland into the incudation medium. Higher dose of synthetic SRIF was needed to produce significant inhibi- tion of GH release from the gland into the medium. Stimulating effects of PGEi on both cyclic AMP formation and GH release in rat anterior pituitary were also suppressed by the addition of synthetic SRIF to the incubation medium. In vivo experiments, synthetic SRIF intravenously injected to the rat showed the inhibitory action to the PGE1 or Na pentobarbital stimulaton of GH release. However, not only the stimulation of GH release and of cyclic AMP production in rat anterior pituitary gland by stimulating substance of GH release, but also the cyclic AMP production induced by synthetic TRH or rat hypothalamic extract were inhibited by SRIF.
These results suggest that synthetic SRIF as well as hypothalamic releasing hormones may act through adenylate cyclase-cyclic AMP system in rat anterior pituitary, although the action of synthetic SRIF seems to be more complicate.

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It has been reported that a myocardial depressant factor (MDF) accumulates in plasma during various types of circulatory shock. In the present study with dogs subjected to cardiogenic shock, MDF was isolated from the plasma on Bio-gel P-2 column chromatography, and determined under the controlled assay condition. MDF was found to be filtrable, soluble in aqueous media, recovered in the chromatographic fraction of molecular weight range between 800 and 1000, heat stable (i. e. 56°C, 30 min), and to give a positive ninhydrin test. The activity of MDF was shown to be markedly increased in parallel with progression of shock states.
In conclusion, MDF is an important determinant in impairment of myocardial performance during circulatory shock.

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The demonstration of the effect of parathyroid hormone (PTH) to stimulate skeletal adenyl cyclase activity in vitro and to enhance cyclic AMP content in young rat calvaria in vivo prompted us to study the cyclic AMP metabolism in rat calvaria correlating to the known hypercalcemic effect of PTH.
Cyclic AMP was extracted from the calvaria dissected from soft tissue in ice cold water and frozen by immersing in dry ice-aceton as described by Chase et al. Competitive binding assay for cyclic AMP in this bone extract was considerably disturbed by an unexplainable interference, so the fraction of the assay responsive to cyclic nucleotide phosphodiesterase was taken as “true cyclic AMP”.
Cyclic AMP content in calvaria of 4 weeks old, thyroparathyroidectomized rat was increased maximally at the end of two minutes infusion of 10 U PTH and returned to control level within 30 minutes (0 time: 0.34, 2 minutes: 2.25, 10 minutes: 0.82, 30 minutes: 0.35 nmoles/g wet tissue). Whether such transient but marked elevation of cyclic AMP content in skeletal tissue was essential for calcium mobilization was examined by studying the response of plasma calcium and cyclic AMP in calvaria to various dose of PTH. Plasma calcium was already increased by infusing 0.5 U of PTH in the absence of detectable change in cyclic AMP metabolism in the skeletal tissue.
Intraperitoneal administration of 10 U of PTH, although raised plasma calcium to comparable extent to intravenous dose, failed to demonstrate any significant elevation of cyclic AMP in calvaria
Hypercalcemic effect of PTH in the absence of change in skeletal cyclic AMP level was also observed in experiment in which EGTA solution was infused into intact rat. Intact rat was recovered from EGTA induced hypocalcemia within one hour, probably due to effective calcium mobilization by endogenous PTH. Even in theophylline pretreated rat, cyclic AMP content in calvaria would not show any significant rise.
The demonstration of hypercalcemic effect of PTH in the absence of change in skeletal cyclic AMP in several experimental conditions might mean the increase in cyclic AMP level required for the hypercalcemic effect of PTH was too small or too transient to be detected by the assay method employed. Any way, it could be concluded that marked increment of cyclic AMP content observed in rat calvaria given relatively high dose of PTH intravenously was not physiological response and was not essential for hypercalcemic effect of PTH.

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Cross innervation studies of fast and slow muscles have shown that motoneurons regulate the speed of contraction, enzyme activities of aerobic and anaerobic glycolysis, calcium uptake of sarcoplasmic reticulum and synthesis of myosin protein. However, mechanism of these regulations is not fully elucidated. Cyclic 3', 5'-AMP (cAMP) is known to regulate the activation of phosphorylase 6 kinase kinase which regulates glycogenolysis and also has many biological activities.
In this study, cAMP and cyclic GMP (cGMP) were measured by the competitive protein binding assays (Gilman, Murad, and Illiano) on white and red muscles.
1) In the state of relaxation, white (fast) muscles contained lower levels of cAMP than those of red (slow) muscles. Pyruvate kinase activity could be regarded as an indicator of differentiation of muscle fibers. Administration of d-tubocurarine caused a significant increase of cAMP in fast muscles.
2) However, adenyl cyclase activities of gastrocnemius and soleus muscles were not different, assayed in the presence of 10 mM NaF.
3) Denervation made by excision of sciatic nerve caused an increase of cAMP/wet weight in gastrocnemius muscles of the rats after 2 weeks.
4) Adenyl cyclase activity of the gastrocnemius muscle from a patient with Duchenne type dystrophy, of which electron microscopic finding revealed only dilated sarcoplasmic reticulum, was lower than controls. Change of cyclase system in the early stage of the dystrophy can be a trigger of the pathologic process.
5) Cyclic GMP was measured after purification on alumina and Dowex 1×8 columns. The concentrations of cGMP of white and red muscles of rabbits were compared with those of cAMP and they showed different levels each other. This suggests that there might exist separate regulatory controls of cAMP and cGMP in skeletal muscle.
Muscle contraction seemed to cause a decrease in cGMP level without change of cAMP.
These results suggest that cAMP may play a role in the so-called trophic influence of nerve on muscle differentiation.

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Fat cells were disrupted in hypotonic medium and lipid micelles were collected by centrifugation. These lipid micelles contained lipase and triglyceride. Incubation of these lipid micelles with adrenaline resulted in marked lipolysis, but only a negligible lipolysis was observed in the absence of this hormone. The lipolytic activity of lipid micelles prepared from isolated fat cells which had been preincubated with 10μg/ml of adrenaline was higher than that of the micelles which preincubated without adrenline and was not stimulated by further addition of adrenaline to the assay system. Lipolysis in the lipid micelles was also increased by phospholipase C, theophylline or CaCl₂ but not by phospholipase D.
On the other hand, no increase of protein kinase was observed during the incubation of the lipid micelles with the lipolytic agents such as adrenaline, phospholipase C, theophylline and CaCl₂.
Based on these results, relationship between mechanism of adrenaline induced lipolysis and cyclic AMP was discussed.

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The activity of glucose 6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) in liver supernatant has been reported to increase both in carbon tetrachloride (CCl₄)-poisoned and high carbohydratefat free diet refed rats. The increase involves the same molecular species of the enzyme. Increased incorporation of ¹⁴C-leucine into G6PD after CCl₄ treatment was not affected by actinomycin D at a dose sufficient to inhibit dietary induction of the enzyme, suggesting that the mechanism of G6PD induction by liver injury appeared to differ in several aspects from that of dietary induction. In this communication, effects of glucagon, cyclic AMP and epinephrine on the level of liver G6PD were tested to obtain further evidences for the difference between these two mechanisms of G6PD induction.
Glucagon, epinephrine plus theophylline and cyclic AMP plus theophylline effectively prevented the induction of liver G6PD following the administration of glucose-casein (7: 3 by weight) to rats after 48 hr fast, although glucagon and epinephrine plus theophylline had no effect on G6PD activity in normal fasted rats. The inhibitory effect by these agents could occur without any change in the amount of diet consumed, suggesting the importance of cyclic AMP concentration in liver in dietary regulation of G6PD level. When cyclic AMP and glucose-casein diet were given simultaneously, there was an inhibition of the rate of enzyme increase depending upon the frequency of the subsequent injection of cyclic AMP, while when cyclic AMP was given later than 12 hr of refeeding, a lag period for G6PD induction, there was no inhibitory effect. This may indicate that some metabolic events occurring in the first 12 hr following the administration of diet would be blocked by cyclic AMP. We have already reported that a glucose prefeeding, which by itself caused no G6PD induction, could eliminate the lag phase as well as the inhibitory effect of actinomycin D on dietary G6PD induction. In the present study, the glucose prefeeding was also found to counteract the inhibitory effect of cyclic AMP on G6PD induction by subsequent glucose-casein refeeding. These results indicated that the inhibitory effect of cyclic AMP could not be observed if a sufficient amount of newly synthesized RNA directed to G6PD induction was already available through a prior glucose feeding. Thus, the effect of cyclic AMP appears to be exerted at the level of transcription.
In contrast to the dietary induction of G6PD, the induction by CCl₄-mediated liver injury was shown to be essentially insensitive to glucagon. This is consistent with already reported facts that liver G6PD induction by CCl₄ liver injury is due to an altered mechanism of posttranscriptional regulation of the enzyme synthesis, probably at the level of translation. It is thus interesting to investigate how altered functions of the polyribosomes in CCl₄-injured liver may participate to G6PD induction. The prior administration of ethyl D-glucofururonoside p-ethoxyphenylhydrazide (CG-DOL)

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A release of cyclic AMP into the bathing medium from the urinary bladder of the toad, Bufo marinus was studied.
Appropriate pieces of the bladder were incubated in 3ml of bicarbonated Ringer's solution for 15 to 60 minutes under different conditions and then the amount of cyclic AMP in the medium and the dry tissue weight of the pieces were measured. The release of the nucleotide was expressed as pmoles/100 mg·dry tissue weight.
Even in the absence of arginine vasopressin (AVP) slight amount of cyclic AMP was found to be released from the tissue predominantly to the serosal medium. During exposure to AVP dose-dependent increases in the release of the nucleotide from the epithelial cells were observed. The maximum effect of 75mU/ml AVP on the release and on the cell level of cyclic AMP appeared nearly 30 minutes after commencement and continued up to one hour.
The release with 75mU/ml AVP was unaffected by 10 mM theophylline, although the cell level showed a further elevation. The recovery of radioactive cyclic AMP added to the bathing medium was also studied under the same conditions. Theophylline was found not to diminish the recovery, suggesting that the agent did not enhance any break-down of cyclic AMP in the medium. These results indicate that theophylline may inhibit the permeation of the nucleotide through the serosal membrane of the epithelial cell into the bathing medium.
Sodium fluoride is known to be a potent stimulator of the activity of adenyl cyclase of cell particle fractions. However, in any in tact cells or tissues the stimulation by sodium fluoride alone has not been reported. In this study a striking elevation of the intracellular cyclic AMP level of the whole cell system, the. intact urinary bladder, was observed with 10 mM sodium fluoride even in the absence of theophylline. This effect exceeded that of 75mU/ml AVP and was accompanied with a simultaneous increase in the release of cyclic AMP and with an increased permeability to water across the membrane of the bladder. These facts suggest that sodium fluoride stimulates the activity of adenyl cyclase even as with the whole cell system.
In brief the epithelial cells of the bladder always release cyclic AMP into the bathing medium predominantly to the serosal side and this release is enhanced by vasopressin and sodium fluoride, but is inhibited by theophylline. It is generally accepted that intracellular concentration of cyclic AMP is determined by the activities of adenyl cyclase and phosphodiesterase. In this study the third determinant, that is, the extent of the release of the nucleotide from the cell was proposed. Some regulatory mechanism for the third process with unknown intrinsic substances should be investigated.

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It is generally accepted that adenosine 3' 5'-cyclic monophosphate (cyclic AMP) is the intracellular mediator of the action of vasopressin in the urinary bladder of the toad. The hormone elevates the concentration of the nucleotide in the epithelial cells of this tissue and exogenous cyclic AMP, as well as the hormone elicits an increase in both net transport of sodium and permeability to water.
Recently it was reported that cyclic GMP stimulates sodium transport in toad bladder, does cyclic AMP, but in contrast to cyclic AMP, does not affect the permeability to water. This study was designed to confirm and extend these observations. Despite considerable effort, we have been unable to demonstrate stimulation of sodium transport by cyclic GMP in a convincing manner.

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Induction of ornithine decarbcxylase by various hormones was investigated in vivo, in slices, dispersed cells and cultured strains of rat liver. The marked induction of the enzyme was observed in vivo 3 hours after injection of either insulin, glucagon, cortisol and cyclic AMP and theophylline. Hydrocortisone showed the best effect for the induction. The enzyme induction was inhibited completely by concomitant injection of either puromycin or actinomycin-D. These inhibitors suppressed the enzyme level far below than that of control. The enzyme level of control experiment increased slightly when rat liver slices were incubated for 4 hours and addition of insulin, glticagon or cyclic AMP enhanced the induction. Dexamethason showed no effect in slices. Similar effect of these hormones were observed in dispersed liver cells.
Induction of this enzyme was also observed in early growing phase of cultured cells (mouse L cell and rat liver B-2 cell) and cyclic AMP inhibited both cell growth and the enzyme induction. These observations were discussed with respect of cell growth.

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The formation of prostaglandin E₁ from bishomo-γ-linolenic acid by the microsomal fraction of bovine vesicular gland, required the simultaneous presence of glutathione, hydroquinone and hemoglobin as reported by other investigators. A new activator which could replace hydroquinone, was isolated and purified approximately 100 fold from the soluble fraction of bovine vesicular gland. This activator was retained upon Sephadex G-200 column chromatography, but eluted faster than hemoglobin. Its activity was lost by heating for 5 min over 60°. Pronase treatment did not affect the activity or the molecular weight of the activator. The inhibition of prostaglandin E₁ synthesis by indomethacin, aspirin and sodium salicylate was observed at lower concentrations in the presence of the activator than in its absence. The activator was adsorbed to Sepharose 4B coupled with indomethacin, and was eluted by increasing the salt concentration.

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Action of prostaglandin (PG) on gastrointestinal motility was studied in vivo and in vitro.
PG-E₁, E₂ and F_2α (10^-7-10^-6M) contracted the isolated longitudinal muscle strips of guinea-pig stomach and colon. However, in contrast to PG-F_2α, PG-₁ and E₂ relaxed the isolated circular muscle strips.
Ca uptake of the circular muscle strip of dog ileum, relaxed in Ca free.KCl solution, was increased with the additon of increasing amount of Ca⁺⁺. At 0.05 mM Ca⁺⁺, Ca-net uptake was 3.13×10^-6 moles/kg wet wt. in the absence of PG-E₁ (10^-5M), but 4.16×10^-6 moles/kg wet wt. in the presence of it. At 0.5 mM Ca⁺⁺, this uptake was 2.89×10^-5 moles/kg wet wt. in control and was 2.49×10^-5 moles/kg wet wt., 1.81×10^-5 moles/kg wet wt. in the presence of PG-E₁ and La⁺⁺⁺ (10^-5M), respectively, at which concentrations Ca contractures were inhibited by 70%.
Propulsive activity of the isolated segmental preparation from guinea-pig colon was increased by the serosal applications of PG-E₁ and F_2α (10^-6M). This increase of the action, however, was abolished in the presence of atropine (5×10^-7g/ml) and tetrodotoxin (10^-7g/ml).
The action of PG-E₁ on gastrointestinal motility in man was investigated roentgenologically. When this decylester (4mg) was orally administered with a barium meal (200ml), intestinal motility was stimulated within 60 min after the meal. However, gastric motility was not.
From these results observed, it was considered that the mechanism of PG-E₁ induced relaxation in circular muscle might depend on the decrease of Ca⁺⁺ influx by a formation of PG-Ca complex within the cell membrane, and that the stimulating action of PGs on intestinal motility in man and the isolated guinea-pig colon might at least exert an indirect effect via the intrinsic nerves within the intestinal wall.

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Nicotinate phosphoribosyltransferase-specific affinity chromatography was prepared from succinylated aminoalkyl agarose coupled with 6-amino nicotinic acid, a competitive inhibitor of nicotinate phosphoribosyltransferase, and was applied to the selective purification of the enzyme of baker's yeast.
Three millimoles of 6-amino nicotinic acid prepared from 6-amino nicotinamide with acid hydrolysis were added to 50ml of succinyldiaminodipropyl agarose at pH 7.4, followed by the addition of three millimoles of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the reaction was allowed to proceed at 4° for 24 hours. The substituted agarose was then washed continuously with distilled water. About 3 μmoles of 6-amino nicotinic acid were covalently bound per ml of agarose. The enzyme solution was added to the affinity column equilibrated with 10 mM Tris-phosphate buffer (pH 8.0) and the enzyme was then eluted with 0.1 M Tris-phosphate buffer containing 0.1 M ammonium sulfate (pH 8.6). About 75-fold purification was attained by this single step procedure. The capacity to adsorb the enzyme to the substituted agarose was strongly affected by the concentration of proteins other than the enzyme.

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Rat liver acetyl-CoA carboxylase was purified by affinity chromatography. An affinity adsorbent was prepared by coupling an inhibitor of liver acetyl-CoA carboxylase, kynurenate, to aminoethyl agarose synthesized from ethylene diamine and CNBr-activated agarose. On chromatography of the ammonium sulfate fraction of rat liver soluble supernatant on a kynurenate-agarose column in the presence of citrate, acetyl-CoA carboxylase was purified about 40 fold with a yield of 90%.

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1. Affinity chromatography was effectively applied to the analysis of complex formation of an enzyme with its specific ligands for the first time.
2. The synthetic procedure for a new support (hydrazide derivative of carboxymethyl-cross-linked dextran) in the preparation of biochemically specific adsorbents is also described. The procedure is based on the hydrazidation of a carboxymethylated polysaccharide via its intra-ester (lactone) derivative.
3. The derived support coupled with some compounds containing primary amino group in a good eff iciency.
4. An analysis of the interaction of carboxypeptidase B with its specific ligands was demonstrated with two kinds of its specific adsorbents which were prepared by the new method.
5. Thq demonstration revealed the multiple ligand-binding sites on the enzyme for small molecular ligands and the cooperative interaction of these sites.

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Affinity chromatography depends on the specific affinity and adsorption of substance for the ligand bound to carrier matrix. Substrate, inhibitor and many other compounds are used as the ligand. In our laboratory, various compounds with low molecular weight and high molecular one were used as ligand, and applied to the affinity chromatography of trypsin, plasmin, plasma kallikrein, thrombin, thymidine kinase and leucine aminopeptidase.
Based on our previous inhibition experiments on trypsin-like enzymes, such as trypsin, plasmin, kallikrein and thrombin, hexyl N^α-(ε-aminocaproyl)-homoargininate was coupled with agarose (Sepharose 4B), and applied to the affinity chromatography of these enzymes. To this agarose column trypsin was adsorbed and eluted with 0.1M acetic acid, although chymotrypsin was not adsorbed. In order to purify plasmin, EDTA-treated euglobulin fraction activated with streptokinase was applied on the agarose column equilibrated with 0.1M borate buffer containing 0.5M NaCl, pH 8.0, washed with the same buffer, and eluted with 0.1M acetic acid. Specific activity was raised from 19 to 108. Because hexyl N^α-(ε-aminocaproyl)-homoargininate has a positively charged guanidino group and hydrophobic group, the agarose coupled with this compound functions as an anion-exchanger, and may adsorb various proteins nonspecifically. In order to avoid such nonspecific adsorption, plasmin solution was applied to the column equilibrated with 0.1M phosphate buffer containing 0.2M NaCl, 10mM benzylamine and 5mM EDTA, pH 7.4, washed with the same buffer, and eluted with 0.1M acetic acid. Specific activity was raised from 14 to 305.
At the same experimental condition, thrombin (Mochida Pharm. Co.) was applied to the agarose column, and eluted with 0.1M acetic acid. Specific activity was raised from 65 to 300. On the other hand, plasma kallikrein activated with acetone was not adsorbed at 0.1M borate buffer containing 0.2M NaCl, pH 8.0, but adsorbed at 0.1M borate buffer. The specific activity was not raised in either low or high ionic strength.
Thymidine kinase preparation, which was prepared by streptomycin treatment and heat treatment at 60° for 3 min of Yoshida sarcoma homogenate was applied on agarose coupled with 5'-amino-5'-deoxythymidine, which is a synthetic inhibitor of thymidine kinase, washed with 0.1M Tris-0.2M NaCl buffer, pH 8.0, and eluted with the same buffer containing 1.5M NaCl. Thus, thymidine kinase was purified 5100 folds from homogenate. The yield was 45 per cent.
In order to purify leucine aminopeptidase from human liver homogenate, leucine amide was used as a ligand. Before application on affinity column, the homogenate was sonicated and digested with bromelain at 37° for 30min. The supernatant was applied on the agarose column at 10mM phosphate buffer, pH 7.5, and eluted with 30mM of the buffer. Because of nonspecific adsorption of various proteins, the raise of specific activity was 4.8-folds.
Since plasmin hydrolyzes fibrinogen, fibrin and casein, EDTA-treated euglobulin fraction activated with streptokinase was applied on fibrinogen-agarose column, and eluted with 0.1M acetic acid. Specific activity was raised to 3.5-folds, however, plasminogen was also adsorbed on this column and eluted as did plasmin. These results indicate that the adsorption of human plasmin on this column does not depend on its specific adsorption.
One molecule of human plasminogen and plasmin combines with one molecule of streptokinase, and forms plasminogen- or plasmin-streptokinase complex. By the adsorption of EDTA-treated human euglobulin fraction on streptokinase-agarose column and elution with 6M urea, highly purified human plasmin, about 70per cent purity, was obtained at 78per cent yield.

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Affinity chromatography was done for identification of the liver collagenase.
According to the Nagai's method, 0.5% acid soluble guinea pig skin collagen was entrapped into 5% acrylamide gel. The gel was ground to 10-50 mesh, and packed into 10×110mm column. The sample was applied through the column with pH 7.5 buffer at 4°, and eluted with pH 5.2 buffer. The recovery of the enzyme activity after affinity chromatography was usually extremely low and varied from 5 to 70%. The part of collagenase activity was passed through with pH 7.5, however, a sharp peak of collagenase activity without detectable protein absorption was recognized with pH 5.2 buffer, and the least amount of caseinolytic activity was determined in this fraction. Some collagenase samples from liver tissue culture medium, liver homogenate reacted with NaSCN, and separated Kupffer cells were purified by affinity chromatography and incubated with collagen. Those enzymes were recognized as collagenase from their mode of reaction by acrylamide disc electrophoresis.
Affinity chromatography was seemed to be useful for detecting tissue collagenase, however, it was not seemed to be adequate to purify a large amount of the enzyme because of its small capacity, The reason for its inconstant recovery was attributed to the character of the gel entrapped collagen.

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Human placental alkaline phosphatase (Alp) was purified by using the immunoadsorbent affinity chromatography.
The commonly used procedures of purifying this enzyme were not only complicated but also time consuming as compared with this method, since the eluate of purified Alp could be easily obtained by only one step without further procedures.
The anti-placental Alp antibody was prepared from a rabbit and coupled with CNBr-Sepharose 4B. The coupled complex was packed in a column. A crude extract of placental Alp was added and washed with 0.2M Tris-HCl buffer (pH 8.0) to exclude contaminated substances. The purified Alp was eluted from the column by 0.2M Na₂CO₃ solution (pH 11.4). A rechromatography of this column was also possible.
The purity of the antigen (=purified Alp made by the previously reported method3) and the specificity of the antigen-antibody reaction are important for the purity of the eluate. To get a large volume of antibody from rabbits is difficult, which causes a limiting scale examination. Those points are to be improved.

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Rabbit antiserum to the purified preparation of adenylate cyclase of Brevibacterium liquefaciens was obtained. A solid phase immunoadsorbent prepared by the cyanogen bromide procedure was shown to bind adenylate cyclase of B. liquefaciens. The elution of bound adenylate cyclase was successfully accomplished with 1 mM to 10 mM NaOH containing 50 mM KCl. Starting from a partially purified preparation, adenylate cyclase was purified 125-fold in the single step of affinity chromatography with a yield of 54%. However, when the partially purified adenylate cyclase was loaded on the immunoadsorbent over the adsorbing capacity, the bound adenylate cyclase was recovered almost quantitatively and approximately 400-fold purification was achieved by this single step procedure. The overall purity was estimated to be 25 to 30% of the homogeneous preparation.
The immunoadsorbent prepared with antiserum to adenylate cyclase of B. liquefaciens was also applicable to the partial purification of adenylate cyclase from Micrococcus lysodeikticus

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Using the polyacrylamide gel disc electrophoresis, the effects of starch on the mobility ofamylases were studied. When the electrophoresis gel contained starch, the mobility of amylases was retarded. The retardation was increased in proportion to starch concentration in the gel. From the change of mobility as a function of starch concentration, the dissociation constants of Taka amylase-A and of EDTA treated human salivary amylase were calculated, to be 2.5×10^-2 g. ml^-1 and 1.9×10^-4 g.ml^-1, respectively. The calculation was based on a theory derived from similar phenomena of chromatography. Therefore, it may also be possible to calculate the dissociation constant of an enzyme from its Rf value on chromatography.

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Lecithin: cholesterol acyltransferase (LCAT) circulates in plasma as an LCAT: high density lipoprotein complex. Plasma high density lipoprotein was fractionated by ultracentrifugation and was coupled to Sepharose 2B according to Porath's technique. This insoluble, but fully hydrated gel was relatively stable to 3M KBr, 0.1 M CaCl₂ or 0.5 mM sodium deoxycholate, however appreciable amount of lipid and protein were dissociated from the gel by exposure to 5 mM sodium deoxycholate.
Partially purified LCAT (equivalent to 40 ml fresh plasma) was applied to a column of HDL-Sepharose (0.9×60 cm), and the column was washed with Tris HCl-EDTA-NaCl buffer, pH 7.4 at 37°C, then the bound LCAT was dissociated with 0.5 mM sodium deoxycholate.The recovery of enzyme was 65 % and the purification was raised by 28 folds by this step. The net purification was approximately 1,500 folds of the original plasma.
HDL-Sepharose which was labelled with 4-C-14 cholesterol was used for the study of enzymesubstrate complex formation and catalytic reaction of LCAT. The inhibition of LCAT reaction by sodium deoxycholate did not run parallel with LCAT: HDL complex formation, and these findings suggest the presence of additional mechanisms for the inhibition of LCAT reaction by sodium deoxycholate.

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Many cases were previously reported about modifications of serum lactic dehydrogenase (LDH) isoenzymes due to complex formation with immunoglobulins. This LDH-binding immunoglobulin is of interest in connection with circulating autoantibodies. However, the exact nature and properties of this unusual immunoglobulin were still not completely elucidated. This report presents immunochemically unique properties of LDH-binding immunoglobulin and describes an application of affinity chromatography to the isolation of immunoglobulin in question.
The antigenic determinants of LDH-binding immunoglobulin were judged by the method of serum immunoelectrophoresis followed by LDH activity staining. In 14 cases studied presently, each of these immunoglobulins had either alpha or gamma of heavy chain (IgA 7, IgG 7). But the type of light chain was proved to be exclusively kappa regardless of differences in heavy chain class. This fact seems to suggest that LDH-binding immunoglobulin in each case is monoclonal and that unique structures responsible for binding to LDH exist in light chain.
Immunoglobulin A, characterized by binding capacity to LDH, was isolated by two steps of affinity chromatographies which were based upon its binding properties to anti-immunoglobulin A and LDH. Methods of insolubilization of legand protein were as follows. CNBr-activated Sepharose 4B (1g) was used as supporting gel and coupled with each legand protein(10mg)under the condition of 0.1M bicarbonate buffer pH 9.0 (0.5M NaCl). After unreacted functioning groups were blocked with 1M ethanolamine, gel was packed in column (gel bed 3.5ml) and washed with 0.1M borate pH 8.0 (1M NaCl) and 0.1M acetate pH 4.0 (1M NaCl) alternately.
At first 1ml of the patient serum was bufferized against 0.2 M glycine-HC1 buffer pH 2.8 (0.5 M NaCl), and filtered on Sephadex G-200 with the same buffer in order to split off LDH. This LDH-free protein fraction was applied on Sepharose 4B coupled with anti-immunoglobulin A and filtered with 0.2 M Tris-HC1 buffer pH 8.0 (0.5M NaCl). IgA fraction obtained by changing of pH of the elution buffer to 2.8 was then labeled with fluorescein isothiocyanate (FITC). Finally FITC-labeled IgA fraction was applied on Sepharose 4B coupled with LDH which was prepared from human hemolysate. By changing of pH a peak of fluorescence intensity corresponding to LDH binding immunoglobulin was observed to elute. The amount of isolated immunoglobulin was just a trace, but the result of present affinity chromatographic experiments indicates that this new specific purification method is effective and applicable to the isolation of various autoantibodies.
In our 14 cases, all of LDH-binding immunoglobulins had exclusively kappa type of light chain.
LDH-binding immunoglobulin A was isolated by the technique of affinity chromatography.

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Affinity chromatography of the calf thymus cysteine-containing histone, rich in arginine and alanine, has been examined basing on the reaction between the SH group and ρ-chloromercuriben-zoated Sepharose. Histories were passed through the column in 5M urea-0.1M citrate buffer, pH 5.5, and the retained fraction was eluted with the denaturant-buffer solution containing 0.05M 2-mercaptoethanol. Further purification of the retained fraction was achieved by Bio-Gel P-60 gel chromatography. The purified fraction showed a single band on polyacrylamide gel electrophoresis and an amino acid in accordance with that obtained by sequence determination.

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