Yuhsi Matuo

Expression of matrilysin in vascular endothelial cells adjacent to matrilysin-producing tumors

Matrilysin is believed to play important roles in tumor progression and metastasis. In the present study, we analyzed matrilysin‐producing cells in various human cancer tissues by immunohistochemistry and in situ hybridization. Tumor cells in colorectal carcinomas, pancreatic carcinomas, transitional‐cell carcinomas of the kidney and small‐cell lung carcinomas were frequently positive for matrilysin. In addition, we found that endothelial cells of arterioles and venules adjacent to matrilysin‐positive tumors expressed matrilysin mRNA and protein. The endothelial cells adjacent to matrilysin‐negative tumors and those in normal tissues were negative for matrilysin. Furthermore, analyses by casein zymography, Western blotting and RT‐PCR showed that matrilysin was weakly expressed by cultured human umbilical vein endothelial cells. Our results suggest that the expression of matrilysin in vascular endothelial cells and in tumor cells may be regulated by common soluble factors, and that endothelial cell‐derived matrilysin may contribute to tumor angiogenesis and tumor metastasis. Int. J. Cancer 72:441–445, 1997.

RAMA stain: A fast, sensitive and less protein-modifying CBB R250 stain

SDS‐PAGE and CBB staining are two of the most popular methods used for protein analysis. Although many reports that describe such staining methods have been published, these conventional protocols require several hours or days for staining and de‐staining. In this study we describe a recently developed, fast and sensitive CBB staining method that utilizes the staining solution of RAMA that consists of the low‐cost reagents: CBB R250, acetic acid, methanol and ammonium sulfate, and the destaining solution of water. Our method dose dependently detects 12 nanograms protein within 60 min and with a wide protein spectrum. Although the features of the dose‐dependent relationship depend upon protein amounts and protein types, for most of the protein samples tested, a linear relationship was observed in the region from 12 to 330u2009ng. Moreover, through further washing, the detection sensitivity of protein is enhanced and reaches a maximum at 1.4u2009ng and then gradually decreases in the de‐staining process. It has been shown recently through MS analyses that the sensitive colloidal CBB staining methods frequently result in artifactual methylations due to the strong acid and long contact during staining and the destaining processes. Such artifacts were reported to be reduced by the replacement of strong inorganic acid with acetic acid and because RAMA utilizes acetic acid and is in contact with the proteins for a short time during staining and de‐staining, it is expected that in vitro artifacts will be reduced. Finally, MS analyses of RAMA‐stained protein bands were revealed not to have been methylated.

Characteristics and applications of N-(ω-aminohexyl--aspartic acid sepharose as an affinity adsorbent☆

1. n1. The affinity adsorbent, N-(ω-aminohexyl)-l-aspartic acid-Sepharose 6B (AHA-Sepharose), was prepared by covalently linking AHA to Sepharose 6B previously activated with CNBr. n n2. n2. The enzymes relating to the metabolism of l-aspartic acid showed a group-specific adsorption to AHA-Sepharose column. Asparaginase, aspartase, aspartate-β-decarboxylase and modified asparaginase with tetranitromethane were adsorbed to AHA Sepharose column and eluted by increasing the ionic strength with NaCl. Asparaginase and aspartase were eluted with the respective substrate or product of lower ionic strength, whereas holo- and apo-enzymes of aspartate β-decarboxylase and the modified asparaginase could not be eluted with the respective substrate or product of lower ionic strength. n n3. n3. Fumarase was not adsorbed to this column. n n4. n4. By the affinity chromatography employing the AHA-Sepharose column, asparaginase from Proteus vulgaris was purified easily and in a high yield.

Novel protease bound with chromatins in normal and tumorous tissues of rats

Abstract A protease capable of hydrolyzing casein with optimum pH 10 (alkaline protease), perhaps functional in hydrolysis of non-histone proteins and Hl histone, was found to exist at the state bound with chromatins of various normal and tumorous tissues of rats, in addition to the protease capable of hydrolyzing histone with optimum pH 8 (neutral protease). Alkaline protease was not observed in other subcellular fractions than nuclear fraction. It had approximately 18,000 daltons, and was chymotrypsin-like as inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor and Chymostatin. Its contents were significantly high in rapidly proliferating cells; Yoshida sarcoma⪢ Rhodamine sarcoma≥ AH 130≥ thymus> spleen⪢ kidney≥ liver⪢ brain.

Cloning and sequencing of the gene encoding the plasma membrane H+-ATPase from an acidophilic red alga, Cyanidium caldarium

A cDNA containing an open reading frame encoding the putative plasma membrane H(+)-ATPase in an acidophilic red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and Southern hybridization based on homologous sequences of P-type ATPases found in other organisms. The cloned cDNA is 3300 bp in length, containing a 2865 bp open reading frame encoding a polypeptide of 955 amino acids which has a predicted molecular mass of 105,371. The deduced amino acid sequence was found to be more homologous to those of P-type H(+)-ATPases from higher plants than that from the green alga Dunaliella bioculata.

Purification and characterization of alkaline protease and neutral protease from chromatin of rats.

It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 000-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.

Purification of coenzyme a by affinity chromatography

Abstract 1. 1. The produced form of coenzyme A (CoA) was immobilized by CNBr-activated Sepharose 6B. By chromatography using the immobilized CoA column, is contained in the dialyzed extracts of the bacteria accumulating CoA. Of the bacteria investigated, Sacrina lutea showed the highest content (approximately 90%) of the protein. 2. 2. The affinity adsorbent, CoA-affinity protein-Sepharose, was prepared by covalently linking the dialyzed extract of Sarcina lutea into Sepharose 6B prebiously activated with CNBr. Reduced CoA, oxidized CoA, dephospho-CoA, ATP and ADP were adsorbed to the adsorbent column, whereas AMP was not. These compounds adsorbed were eluted by increasing ionic strength (I) with NaCl. When the column was equilibrated with sodium acetate buffer (pH 6.0, I=0.04), reduced CoA was selectively adsorbed. 3. 3. By affinity chromatography employing CoA-affinity protein-Sepharose, reduced CoA pf 92% purity was obtained in a yield of 94% from crude reduced CoA of 5% purity. 4. 4. The adsorbing capacity of the adsorbent to reduced CoA was 85 μg per ml of the adsorbent under operating conditions at pH 6–7 and 10–25°C. The capacity of the adsorbent was not changed by stroge at 5°C and pH 6.0 for a month.

PREPARATION OF IMMOBILIZED TANNINS FOR PROTEIN ADSORPTION

Preparation and adsorption specificity of tannins immobilized by covalent binding on water-insoluble matrices were investigated. Immobilized tannins were prepared by condensing cyanogen bromide activated tannins with aminohexyl derivatives of several kinds of matrices. The most suitable matrix for the immobilization of tannin was alkali-treated cellulose powder. The concentration of sodium hydroxide solution for alkali treatment influenced the adsorption capacity of immobilized tannin for a protein, but temperature and time for alkali treatment did not. Immobilized tannins having spacers of long chain length exhibited high adsorption capacity for a protein. Chinese gallotannin was the most favorable ligand for protein adsorption. The immobilization of tannin on aminohexyl matrices was also possible by using epichlorohydrin instead of cyanogen bromide. The maximum adsorption capacity of the immobilized tannin for a protein was about 50 mg/ml of the absorbent. Immobilized tannin adsorbed proteins specifically but did not absorb low molecular weight compounds.

Purification of flavin-adenine dinucleotide and coenzyme a on p-acetoxymercurianiline-agarose columns☆

1. n1. The adsorbent p-acetoxymercurianiline-Sepharose 6B (PAMA-Sepharose 6B) was prepared by covalently linking PAMA to CNBr-activated Sepharose 6B. n n2. n2. When the adsorbent column was equilibrated with dilute acetic acid (ionic strength, Γ2 = 0.005, pH 3–5) FAD, FMN, ATP or ADP was found to be adsorbed to the column, whereas riboflavin, AMP and adenosine were not. The adsorbed compounds could be eluted with 0.1 m NaCl. The adsorbing capacities to these compounds were maximal at pH 4.0. n n3. n3. When the adsorbent column was treated with 1% 2-mercaptoethanol (2-ME) and then equilibrated with the buffer (Γ2 = 0.1–1.0, pH 3–7), reduced CoA, cysteine and glutathione were adsorbed to the column, whereas oxidized CoA, FAD, ATP, ADP and AMP were not. These adsorbed compounds could be eluted with 1% 2-ME. If the adsorbent column was not previously treated with 1% 2-ME, these sulfhydryl compounds were adsorbed to the column in an irreversible manner. n n4. n4. By chromatographic purification employing the adsorbent column at Γ2 = 0.005 and pH 4.0, FAD of 98% purity was isolated in a yield of 95% from a partially purified sample of 75% purity. n n5. n5. By chromatographic purification employing the adsorbent column at Γ2 = 0.1 and pH 5.0, reduced CoA of 95% purity was obtained in a yield of 90% from a crude sample of 5% purity. n n6. n6. The adsorbing capacity for FAD was 1.2 mg per ml of the adsorbent under operating conditions of Γ2 = 0.005 and pH 4.0, and that for reduced CoA was 3.9 mg per ml of the adsorbent at Γ2 = 0.1 and pH 5.0. The capacities of the adsorbent for both compounds were not changed by storage at 5°C in acetone for at least 2 months.

Difference in physical and chemical properties between muscle-type and spleen-type isoenzymes of pyruvate kinase in rat.

Abstract Muscle(M)-type and spleen(S)-type isoenzymes of pyruvate kinase were prepared to a homogeneous purity from skeletal muscles and the tumor (Rhodamine sarcoma) of rats, respectively. The respective types were composed of four of the identical subunit of approximately 60,000 daltons. Their amino acid compositions and peptide maps resembled each other, although there were some definite differences. The total number of dicarboxylic and basic amino acids were higher and lower with M-type than with S-type, respectively, in accordance with the difference in isoelectric point. Of the ninhydrin-positive spots on the peptide maps of their tryptic digests, 61 spots were common for both types, whereas 4 spots were specific to M-type and 5 spots to S-type.