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J Tang

Publications and source records attributed to J Tang.

At least 613 records · Page 34Linked to original sources
PubMed1979

Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements.

Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.

Amino Acid Sequence↗
PubMed1979

Evolution in the structure and function of carboxyl proteases.

A model for the structure and function of extracellular carboxyl (acid) proteases can be established from three amino acid sequences and four crystal structures of these enzymes. The carboxyl proteases from gastric and fungal origins are very homologous in both primary and tertiary structures. The molecules consist of about 320 residues organized with a secondary structure which is primarily comprised of beta-strands and very similar tertiary structures. An apparent binding cleft, which can accommodate a substrate with about eight amino acid residues, contains near its midpoint the active center residues Asp-215, Asp-32, and Ser-35. These three residues are hydrogen bonded to each other. An intracellular carboxyl protease, cathepsin D, is very homologous to the extracellular enzymes in N-terminal amino acid sequence and primary structure location of active center residues. The tertiary structure of cathepsin D is probably similar, as well. However, cathepsin D contains a unique hydrophobic "tail" made up of about 100 residues added on the C-terminal side. Cathepsin D precursor is over 100,000 daltons in molecular weights, as contrasted to the gastric carboxyl protease zymogens, which are about 40,000 daltons. Carboxyl proteases contain two lobes symmetrical in peptide chain conformations. Each of the lobes also consists of two homologous structural units. These structural characteristics suggest that the original gene was coded for only about eighty amino acid residues and that gene duplication and fusion has taken place twice to produce a single chain carboxyl protease with four basic structural units in two symmetrical lobes. The formation of the zymogens and the cathepsin D "tail" must have resulted from various gene fusions. Partial sequence comparisons also suggest that cathepsin D may be an evolutionary ancestral chain for gastric carboxyl proteases.

Amino Acid Sequence↗
PubMed1979

Structure of the lipopolysaccharide from an Escherichia coli heptose-less mutant. I. Chemical degradations and identification of products.

The structure of lipopolysaccharide from a heptose-less mutant of Escherichia coli K-12 has been investigated. Lipopolysaccharide isolated from 32P-labeled cells was treated with mild alkali to yield two separable components: [OH-LPS]-I (approximately 70%) and [OH-LPS]-II (approximately 30%). Mild acidic treatment of [OH-LPS]-I gave mainly a product which was identified as (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine 1-phosphate (Compound I). Further acidic hydrolysis of both [OH-LPS]-I and [OH-LPS]-II yielded as the main product (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine (Compound II). The structures of the above products were deduced by a combination of compositional analyses, sensitivity to phosphomonoesterase, rates of hydrolysis of the phosphate groups and alkali-catalyzed beta elimination of the phosphate residues following appropriate oxidation of hydroxyl groups. These studies together with work reported in the accompanying papers have led to the identification of two species of lipopolysaccharide in the E. coli strain both of which contain a single glucosamine dissacharide unit but differ in having monosubstituted phosphate or pyrophosphate groups at the glycosidic position. Each species of lipopolysaccharide also appeared to be heterogeneous with respect to the number of esterified fatty acyl groups.

Alkaline Phosphatase↗
PubMed1978

Structural evidence for gene duplication in the evolution of the acid proteases.

X-ray studies of acid proteases indicate a bilobal structure with a well defined active site cleft. An intramolecular twofold symmetry axis relates two topologically similar domains and the active site residues. A possible mechanism for evolution by gene duplication, divergence and gene fusion is presented.

Amino Acid Sequence↗
PubMed1977

Pepstatin inhibition mechanism.

Pepstatin is a low molecular weight, potent inhibitor specific for acid proteases with a Ki value of about 10(-10)M for pepsin. The chemical structure of pepstatin is essentially a hexapeptide which contains two residues of an unusual amino acid, 4-amino-3-hydroxy-6-methylheptanoic acid (statine). The complete structure of pepstatin is isovaleryl-L-valyl-L-valyl-statyl-L-alanyl-statine. To study its mode of inhibition, we prepared several derivatives and measured their kinetics of inhibition. Both N-acetyl-statine and N-acetyl-alanyl-statine are competitive inhibitors for pepsin with Ki values of 1.2 x 10(-4)M and 5.65 x 10(-6)M, respectively. The Ki value for N-acetyl-valyl-statine is 4.8 x 10(-6)M. These statyl derivatives, therefore, are very strong inhibitors. The Ki value for N-acetyl-statine is 600-fold smaller than that of its structural analog N-acetyl-leucine. The derivative which contains two statyl residues in a tetrapeptide exhibits inhibitory properties which approach those of pepstatin itself. Other acid proteases, human pepsin, human gastricsin, renin, cathepsin D, the acid protease from R. chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. We suggest that the statyl residue is responsible for the unusual inhibitory capability of pepstatin and that statine is an analog of the previously proposed transition state for catalysis by pepsin and other acid proteases.

Animals↗
PubMed1977

Intramolecular activation of pepsinogen.

Two pathways for pepsinogen activation have been demonstrated. Intramolecular activation, which is kinetically first-order, predominates over the autocatalytic pathway if the pH is below 3 and the protein concentration is less than 1 mg/ml during activation. Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta.

Animals↗
PubMed1976

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.

Amino Acids↗
PubMed1976

Mode of inhibition of acid proteases by pepstatin.

Four derivatives of pepstatin, each of which contains the unusual amino acid 4-amino-3-hydroxy-6-methylheptanoic acid (statine) have been prepared. All four are porcine pepsin inhibitors. Both N-acetylstatine and N-acetyl-alanyl-statine are competitive inhibitors for pepsin with Ki values of 1.2 X 10(-4) M and 5.65 X 10(-6) M, respectively. The Ki values for N-acetyl-valyl-statine is 4.8 X 10(-6) M. These statyl derivatives, therefore, are very strong inhibitors. The Ki value for N-acetyl-statine is 600-fold smaller than that of its structural analog N-acetyl-leucine. The derivative which contains two statyl residues in a tetrapeptide exhibits inhibitory properties which approach those of pepstatin itself. Other acid proteases, human pepsin, human gastricsin, renin, cathepsin D, the acid protease from Rhizopus chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. It is suggested that the statyl residue is responsible for the unusual inhibitory capability of pepstatin and that statine is an analog of the previously proposed transition state for catalysis by pepsin and other acid proteases.

Amino Acid Sequence↗
PubMed1976

Purification and properties of cathepsin D from porcine spleen.

Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species.

Amino Acid Sequence↗