Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.     

Inhibition of platelet aggregation by S-(1,2-dicarboxyethyl)glutathione, intrinsic tripeptide in liver, heart, and lens

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or baker's yeast showed strong inhibitory effects on blood coagulation and platelet aggregation. The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate. DCE-GS did not show chelating activity. As for ADP- or thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation. DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen. The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol-13-acetate. Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different.     

Stereospecific Reduction of Ethyl 2′ -Ketopantothenate to Ethyl D-(+)-Pantothenate with Microbial Cells as a Catalyst

A novel enzymatic process for the synthesis of D-(+)-pantothenic acid through the asymmetric reduction of the 2′ -ketopantothenate ester is described. Candida macedoniensis AKU 4588 was found to convert ethyl 2′ -ketopantothenate (80 mg/ml) almost specifically to ethyl D-(+)-pantothenate (>98% enantiomeric excess), with a molar yield of 97.2%.     

Interrelationship between serum muscle enzymes and a low T3 in anorexia nervosa

To determine the interrelationship between muscle dysfunction and a low T3 state, both seen in anorexia nervosa, we studied the relationship between the degree of muscle involvement, as assessed by the circulating concentration of the three muscle indicators (CPK, GOT and LDH), and serum T3 in thirty-three patients when they were admitted to the hospital. We also studied the malnutritional state, as assessed by their body weight or serum GH, serum potassium and the degree of hyperactivity exhibited. Additionally, another twelve patients were studied in order to explore the mounding phenomenon which is typically elicited in hypothyroidism. The logarithms of serum CPK and GOT correlated only with the serum T3 concentration (r = -0.35, p less than 0.05; r = -0.41, p less than 0.05; respectively). The logarithm of serum LDH highly correlated with serum T3, the percentage of ideal body weight, and the logarithm of serum GH (r = -0.55, p less than 0.01; r = -0.66, p less than 0.001; r = 0.43, p less than 0.05; respectively). The mounding phenomenon was elicited in ten out of twelve patients. In conclusion, it was implied that a low T3 state was associated with an increase in serum muscle indicators and thus with muscle dysfunction encountered in anorexia nervosa.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

Nucleotide sequence analysis of the complement resistance gene from plasmid R100

The multiple antibiotic resistance plasmid R100 renders Escherichia coli resistant to the bactericidal action of serum complement. We constructed a plasmid (pOW3) consisting of a 1,900-base-pair-long restriction fragment from R100 joined to a 2,900-base-pair-long fragment of pBR322 carrying ampicillin resistance. E. coli strains carrying pOW3 or R100 were up to 10,000-fold less sensitive to killing by serum complement than were plasmid-free bacteria or bacteria carrying pBR322. Nucleotide sequencing revealed that 875 of the 1,900 bases from R100 correspond exactly to part of the bacterial insertion sequence IS2. The remaining 1,075 bases contained only one sizeable open reading frame; it covered 729 base pairs (243 amino acids) and was preceded by nucleotide sequences characteristic of bacterial promoters and ribosome binding sites. The first 20 amino acids of the predicted protein showed features characteristic of a signal sequence. The remainder of the predicted protein showed an amino acid composition almost identical with that determined for the traT protein from the E. coli F factor. Southern blot analysis showed that the resistance gene from R100 does not hybridize to the serum resistance gene from ColV,I-K94 isolated by Binns et al.; we concluded that these genes are distinct.     

Increase in Alkaline Phosphatase Activity in Cucumber Roots during Calcium Starvation

Alkaline phosphatase activity in cucumber roots increased withcalcium deficiency. However, acid phosphatase was scarcely affectedby the treatment. Re-addition of calcium not only suppressedthe continuous increase in alkaline phosphatase activity butalso reduced the already formed activity. Alkaline phosphatase activity increased with a deficiency ofcalcium, phosphate, minor elements and iron in this order. Eithercycloheximide or actinomycin D completely suppressed the increasein the activity caused by calcium deficiency. (Received April 16, 1981; Accepted July 17, 1981)     

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide by 3-chloro-D-alanine hydrogen chloride-lyase (deaminating) of Pseudomonasputida

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide is catalyzed by highly purified 3-chloro-D-alanine hydrogen chloride-lyase from $\text{Pseudomonas}\text{putida}$. The synthetic reaction proceeds optimally at pH 8.5, as a function of enzyme concentration and incubation time. The enzymatically synthesized D-cysteine was isolated from the large scale reaction mixture and identified by physicochemical means.     

Refractoriness to phosphaturic action of parathyroid hormone occurring independent of phosphate depletion in hyperparathyroid rats

In an attempt to clarify the underlying mechanism(s) in the disappearance of phosphaturic response to bolus parathyroid hormone (PTH) in hyperparathyroid patients, the effects of bolus bovine PTH (10 USP U) were studied in conscious thyroparathyroidectomized (T . PTX) male Wistar rats that had been infused with a dose of PTH (2.5 U/hr, for 16 hours) so as to reproduce hyperparathyroidism. These animals responded with an increase in urinary cyclic AMP, but without an increase in renal clearance of phosphate. The loss of phosphaturic response was not prevented by pretreatment with actinomycin D at a dosage close to full toxicity (0.1 mg/kg BW, ip, for 3 days). Actinomycin D at this dosage did not affect the normal stimulatory effects of bolus PTH on urinary cyclic AMP and renal clearance of phosphate in T . PTX rats. The continuous infusion of PTH produced nearly maximal phosphaturia throughout in the face of a significant depletion of phosphate. In addition, pretreatment with actinomycin D did not cause a further increase in urinary phosphate excretion during the infusion. These results, along with the report of Shah et al. (1979) indicating that the development of antiphosphaturic adaptation to acute phosphate depletion was prevented by comparable amounts of actinomycin D, indicate that the disappearance of phosphaturic response to bolus PTH by prior PTH infusion simply signifies the continuation of maximal phosphaturic response to the preceding PTH infusion. It is also suggested that the continuous action of PTH prevents, at least phenomenologically, the development of the gene-activation-mediated refractoriness to PTH or antiphosphaturia induced by acute phosphate depletion.     

Presence of a thiol protease in regenerating rat-liver nuclei. Partial purification and some properties

1. Nuclei of regenerating rat liver washed with Triton X-100 were found to contain a new protease. Since the enzymatic activity for degrading ribosomal proteins was inhibited in vivo by administration of E-64, a thiol protease inhibitor, the enzyme may participate in the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver nuclei as reported previously by us [Biochem. Biophys. Res. Commun. 75, 525-531 (1077)]. The optimum pH was 5.5. 2. The enzyme was extracted from washed nuclei and partially purified by gel filtration through Sepharose 6B. Its molecular weight was about 40 000. A maximal activity of partially purified enzyme was observed in the presence of 1 mM EDTA and 2 mM dithiothreitol at pH 5.5 It was inhibited by thio reagents, E-64, leupeptin and hevy metal ions. The enzyme degraded ribosomal proteins endoproteolytically and degraded most proteins tested as substrates, although liver cell sap proteins and serum albumin were less degraded than ribosomal proteins and histones, alpha-N-Benzoylarginine-beta-naphthylamide and benzoylarginine amide were not hydrolyzed.