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Biomedical subjects

V Turk

Publications and source records attributed to V Turk.

At least 19 recordsLinked to original sources
PubMed1992

Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding.

Thermal denaturation of two homologous proteins, low-M(r) cysteine-proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65 degrees C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half-denaturation at 50.2 degrees C and 90.8 degrees C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively. Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three-dimensional structure has been determined. The calculated enthalpy, heat-capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.

Calorimetry, Differential Scanning↗
PubMed1992

Intermediates in denaturation of a small globular protein, recombinant human stefin B.

Guanidinium HCl (GdmHCl), pH, and heat denaturation of the recombinant human stefin B, a low molecular weight protein inhibitor of cysteine proteinases, has been followed by circular dichroism. From the noncoincidence of the transitions in the near and far UV, the existence of stable intermediate states possessing few persistent tertiary interactions but most of the native-like secondary structure, was inferred. These intermediate states exist at equilibrium under various conditions, namely, state G at 1.7 M GdmHCl (pH 8, 25 degrees C), state A at pH 4 (0.6 M GdmHCl, 25 degrees C) and state T above 68 degrees C. By size exclusion chromatography, their apparent compactness was determined. The intermediate states A, T, and G were compact and are therefore classified as "molten globule" states.

Chromatography, Gel↗
PubMed1992

The complete primary structure of bovine stefin B.

A new stefin B-type low-Mr CPI was isolated from bovine thymus and subjected to structural analysis. The inhibitor consisted of 98 amino acids and its Mr was calculated to be 11,178. The NH2-terminal amino acid residue was blocked. The sequence was determined by automated sequencing of peptides derived by cleavage with cyanogen bromide and fragments of the inhibitor resulting from enzymatic digestion with beta-trypsin and Staphylococcus aureus V-8 proteinase. The NH2-terminal blocking group was established with mass spectrometry. The inhibitor exhibits considerable sequence homology with inhibitors from the stefin family. Furthermore, a highly conserved QVVAG region within the stefin family is for the first time replaced by the QLVAG sequence.

Amino Acid Sequence↗
PubMed1992

Cystatins and stefins in ascites fluid from ovarian carcinoma.

Cysteine proteinase inhibitors (CpI) of all three families were found in ascites fluid from patients with ovarian carcinoma. CPIs were isolated by affinity chromatography on carboxymethylated papain Sepharose, followed by gel filtration, anti-stefin-Sepharose and ion exchange chromatography. The highest apparent inhibition against cathepsin B (Cat B) was found in the low molecular mass (LMM) CPI fraction. Immunochemical analysis of this fraction revealed the presence of cystatin C and both stefins A and B while the high molecular mass (HMM) CPI fraction contained kininogens. We demonstrated that CPIs were not completely associated with cysteine proteinases (CPs): about 20% of HMM CPIs and 50% of LMM CPIs were free in native ascites fluid. Affinity chromatography on anti-Cat B-Sepharose revealed that the major LMM CPI, associated with Cat B in native ascites fluid, was the full length form of cystatin C, pI 9.3, and not its truncated form, pI 7.85. The latter was isolated and found to inhibit Cat B in vitro with apparent Ki 0.18 +/- 0.2 nM. Stefin A was isolated from alkaline activated ascites fluid in its two isoforms, pI 4.6 and 4.9. In native ascites, the pI 4.9 isoform was mostly associated with Cat B. Ki for Cat B was 3.55 +/- 1.7 nM, not significantly different from the Ki values measured for stefin A, isolated from other human tissues and biological fluids.

Adult↗
PubMed1992

Stefins and lysosomal cathepsins B, L and D in human breast carcinoma.

In the study of 50 matched pairs of breast carcinoma and normal breast tissue, the activities of cysteine proteinases (CPs), cathepsin (Cat) B and Cat L in tumors were increased on average by 18.5-fold and 52.5-fold respectively. The differences in activity of cysteine proteinase inhibitors (CPIs) between tumor and control breast tissues was also observed: in approximately two thirds of carcinomas, lowered CPI activity was measured (group-I patients), while similar or higher tumor CPI activity was measured in the remaining samples (group-II patients). Relative increases in specific activity of Cat B and Cat L in group I were significantly higher than in group II. In group I more patients with histopathological tumor grade III and negative estrogen (ER) and progesterone receptor (PR) levels were found, but the metastatic involvement of regional lymph nodes was similar in both groups. A 2-year follow-up study showed a significant inverse correlation between disease-free survival and increased Cat L activity, but the differences in group I and group II patients were not significant in this short time interval. In 20 matched pairs of breast carcinoma and normal breast tissue, the mean activity of Cat D was 5.8-fold higher in tumors compared with controls. The hypothesis that elevated Cat D activity increased CP activity and/or lowered tumor CPI activity due to post-translational proteolytic modification appeared less likely, since no correlations between corresponding activities were observed. We suggested that lowered CPI might rather reflect changes in transcription of intracellular CPIs, the stefins. Immunoassay and Northern blot analysis showed that the average value of stefin A protein and mRNA content respectively in the majority of investigated breast carcinoma samples were lowered, suggesting the possible value of stefin A in diagnosis and/or prognosis of the disease.

Blotting, Northern↗
PubMed1992

Isolation and characterisation of chicken L- and H-kininogens and their interaction with chicken cysteine proteinases and papain.

We have isolated L- and H-kininogens from chicken egg white and plasma using affinity chromatography on CM-papain Sepharose, Con-A Sepharose, gel filtration and ion exchange chromatography, in pure form. In chicken plasma mostly HK was present, whereas in chicken egg white both LK and HK were identified. The determined Mr of LK and HK were 69000 and about 96000, respectively. They occurred in multiple forms with pI 4.3-5.2. The inhibitory activity of chicken HK was tested against cysteine proteinases cathepsins B and L, isolated from chicken liver, and papain. The obtained Ki values demonstrated that chicken H-kininogen is a strong inhibitor of chicken cathepsin L and papain, but much weaker inhibitor of chicken cathepsin B.

Amino Acid Sequence↗
PubMed1992

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.).

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5'-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.

Amino Acid Sequence↗
PubMed1992

Bovine cathepsins S and L: isolation and amino acid sequences.

The purification procedure of cathepsin S includes acid activation of spleen homogenate, incubation at 37 degrees C, precipitation with (NH4)2SO4 in H2O/tert-butanol medium, gel chromatography, chromatofocusing, covalent chromatography and cation chromatography of FPLC system. Cathepsin S has a M(r) of about 24,000 Da with pI of 6.5 and 6.8. The mixture of both forms gave a single sequence. Cathepsin L was purified from bovine kidney by acid treatment and incubation of 37 degrees C, precipitation by (NH4)2SO4, two ion exchange chromatographies on CM-Sephadex, gel chromatography and ion exchange chromatography on FPLC system. Cathepsin L exists in multiple forms with pI 5.3-5.7 and M(r) of about 29,000 Da. N-terminal amino acid sequence confirms that cathepsin L and cathepsin S are different enzymes.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of bovine stefin B.

A new cysteine proteinase inhibitor (CPI) was isolated from bovine thymus. According to the amino acid sequence it belongs to the stefin family. It appears as a monomer and a dimer with monomer M(r) of 11,178 and pI values 5.6 for the monomer and 5.2 and 5.6 for the dimer. Ki for the interaction with papain was determined to be 0.12 nM. The most interesting feature of bovine stefin B is the replacement of the highly conserved QVVAG region in stefins with the QLVAG sequence without interfering its inhibitory properties.

Amino Acid Sequence↗
PubMed1992

Denaturation of stefin B by GuHCl, pH and heat; evidence for molten globule intermediates.

GuHCl, pH and thermal denaturation of the recombinant stefin B was followed by circular dichroism (CD) and size-exclusion chromatography (SEC). CD at 277 nm was taken as an indicator of integral tertiary structure and CD at 222 nm as an indicator of the secondary structure. Compactness was expressed by the volumes of elution on a SEC (Superose 12) column. Data on equilibrium denaturation were recalculated to the fractions of the native state (fN). The results have shown that equilibrium intermediates of the molten globule type exist under conditions of low pH, high temperature or medium GuHCl concentrations, namely A, T and G. Recent findings on structure and energetics of molten globule intermediates are reviewed.

Circular Dichroism↗
PubMed1992

Characterization and structure of pineapple stem inhibitor of cysteine proteinases.

The complete amino acid sequence of the inhibitor of cysteine proteinases from pineapple stem acetone powder was determined. The inhibitor consists of 52 amino acids and is composed of two polypeptide chains (41 and 11 amino acids) linked via disulphide bonds. It differs from already known sequences in one to four amino acids. Data from its amino acid sequence analysis clearly show that this inhibitor cannot be a member of the cystatin superfamily. The Ki values for papain, bromelain and cathepsin L were determined.

Amino Acid Sequence↗
PubMed1992

Cystatins and cathepsins in breast carcinoma.

The increased expression of proteolytic systems is one of the characteristics of transformed and malignant cells and their evaluations in whole tumor homogenates were considered as possible diagnostic and/or prognostic factors. Abnormal intracellular distribution, increased activities and secretion of cysteine proteinases (CPs) cathepsin B (Cat B) and L (Cat L), were associated with tumor progression. In the present study of matched pairs of breast carcinoma and normal breast tissue, the activities of Cat B and Cat L in breast carcinoma homogenates were found to be 20 and 50 fold higher, respectively, than in normal tissues. In contrast, a decrease in total inhibitory activity of cysteine proteinase inhibitors (CPIs) was observed but an average ratio between tumor and normal tissues was only 0.75. One of the CPIs, stefin A, was also determined immunochemically. The activities of CPs and CPIs were compared to the increased levels of cathepsin D (Cat D) activities in individual patients, but no statistically significant correlations were found. We correlated CPs and CPIs with morphological and receptor data as well as the axillary lymph node metastases. There was no statistical correlation of CP and CPIs with the number of lymph node metastases. However, highly elevated levels of Cat B and Cat L and lowered CPI activities in tumor cytosols were often associated with poorly differentiated carcinomas and those with negative ER and PR values. We conclude that cysteine-dependent proteolysis may play an important role in breast tumors.

Amino Acid Sequence↗
PubMed1992

Cathepsins B, H and L in human breast carcinoma.

We have measured the activity of the cysteine proteinase cathepsin B, the inhibitory activity of cysteine proteinase inhibitors and the amounts of cathepsins B, H and L in normal sera and sera from patients with breast cancer, as well as in tissue homogenates of tumorous and non-tumorous samples. The amounts of cathepsin B, determined by ELISA in tumour sera (n = 17) were significantly higher than the amounts determined in normal sera (n = 20). On the other hand, the differences in the amounts measured in tumour sera were not significant when compared with the known histopathological characteristics. In cytosols of breast cancer tumour tissue in general, the level of cathepsin H was higher than those of cathepsins B and L in all samples tested. In the same samples, at least a 10-fold increase of cathepsin B (activity and quantity) was detected in matched pairs (n = 20) of carcinoma and normal tissue of the same breast (p less than 0.01). The amount of cathepsin B correlated with the degree of malignancy inside the histological subtypes of invasive ductal carcinoma (n = 90, p less than 0.01). In addition, a negative correlation of values for cathepsin B with the involvement of regional lymph nodes (n = 75, p less than 0.01) was found inside the same group. In contrast, the activities of cysteine proteinase inhibitors did not correlate with any of the known clinical data. Our data provide further indirect evidence for the involvement of cathepsin B in the processes of tumour growth and metastasis in breast carcinoma. The follow-up studies will verify the prognostic value of these findings.

Breast Neoplasms↗
PubMed1992

Determination of cathepsins B, H, L and kininogen in breast cancer patients.

In 20 matched pairs, an increase of all cathepsins was measured in carcinoma tissue compared with normal tissue of the same breast. On contrary, the mean value for kininogen was almost 2 fold lower than mean value determined in normal cytosols. From all proteins tested, the amount of cathepsin B correlated mostly with the degree of malignancy inside the group of invasive ductal carcinoma (n = 90, p < 0.01).

Biomarkers, Tumor↗
PubMed1991

The cystatins: protein inhibitors of cysteine proteinases.

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.

Amino Acid Sequence↗
PubMed1991

Folding studies of the cysteine proteinase inhibitor--human stefin A.

Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.

Chromatography, High Pressure Liquid↗