Sequence position of 3-hydroxyproline in basement membrane collagen. Isolation of glycyl-3-hydroxyprolyl-4-hydroxyproline from swine kidney.

The position of 3-hydroxyproline was investigated in the triplet sequences of peptides released by collagenase digestion of a collagen preparation from kidney cortex. Composition of the collagen preparation indicated that it was largely or wholly of basement membrane origin. 3-Hydroxyproline was detected in only one sequence, the tripeptide, glycyl-3-hydroxyprolyl-4-hydroxyproline, which accounted for a major fraction of the total 3-hydroxyproline obtained in the peptides released by collagenase. Preliminary data, based on sequencing the peptide mixture released by collagenase treatment, suggested that, in contrast, 4-hydroxyproline occurs predominantly if not exclusively in the Y position of Gly-X-Y triplet sequences in the collagen preparation studied.     

Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

A new rapid method for quantitating radioactive proline, 4-hydroxyproline,and 3-hydroxyproline

A simple and rapid method for separating proline, 4-hydroxyproline and 3-hydroxyproline was developed by the use of high-voltage electrophoresis. An excellent counting efficiency of proline was achieved by a simple extraction of the labeled material from paper; 98–100% recovery of count rates was obtained, equivalent to recoveries from column chromatography. This method can tolerate high concentrations of salt, acid, and protein in the sample. No carrier is required and multiple samples (up to 15) can be separated on a single sheet and analyzed within 2 h. Serial dilution experiments showed excellent linearity. An average recovery rate of 92% was obtained for samples over a wide range of radioactivity and high sensitivity of the method was demonstrated. This analysis is applicable to protein hydrolysates and to determination of the free amino acids in the presence of protein. Thus, proline, 4-hydroxyproline and 3-hydroxyproline can be quantitated simultaneously in any biological sample.     

Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product.

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.     

The synthesis of macromolecular 3-hydroxyproline by attaching and confluent cultures of human fibroblasts.

The synthesis of collagen has been studied during the attachment of freshly trypsinized human fibroblasts to culture vessels by measurement of the incorporation of radioactive proline into macromolecular hydroxyproline. Collagenous protein(s) was found to be a component of a substrate-attached material ('microexudate carpet') synthesized rapidly during cell attachment in the absence of serum. The ratio of 3-hydroxyproline/4-hydroxyproline in the collagenous proteins synthesized during cell attachment was found to be 4-5 fold higher than that of normal type I collagen. The synthesis of 3-hydroxyproline by confluent cultures was diminished by serum deprivation, and was shown to require higher concentrations of ascorbate than the synthesis of the 4-hydroxy isomer.     

Attaching human fibroblasts secrete a type I procollagen rich in 3-hydroxyproline.

The predominant collagenous protein secreted during the attachment of freshly trypsinized human foreskin fibroblasts was found to be Type I procollagen. Evidence is presented that both the α₁ and α₂ chains exhibit a 3-hydroxyproline/4-hydroxyproline ratio 4–5 fold higher than that of normal Type I collagen. These findings suggest that caution should be exercised in assigning an observed increase in the 3-hydroxyproline/4-hydroxyproline ratio to the synthesis of a basement membrane type collagen.     

Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.     

Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates.

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.     

Increased molecular damage and heterogeneity as the basis of aging

Aging at the molecular level is characterized by the progressive accumulation of molecular damage. The sources of damage act randomly through environmental and metabolically generated free radicals, through spontaneous errors in biochemical reactions, and through nutritional components. However, damage to a macromolecule may depend on its structure, localization and interactions with other macromolecules. Damage to the maintenance and repair pathways comprising homeodynamic machinery leads to age-related failure of homeodynamics, increased molecular heterogeneity, altered cellular functioning, reduced stress tolerance, diseases and ultimate death. Novel approaches for testing and developing effective means of intervention, prevention and modulation of aging involve means to minimize the occurrence and accumulation of molecular damage. Mild stress-induced hormesis by physical, biological and nutritional methods, including hormetins, represents a promising strategy for achieving healthy aging and for preventing age-related diseases.     

Benzodiazepine receptor heterogeneity: possible molecular basis and functional significance

The pharmacological actions of the benzodiazepines (BZs) are thought to be mediated through specific receptor sites in the mammalian central nervous system. Characterization of these receptor sites in the brain has yielded evidence for heterogeneity of BZ receptor sites. Current theories on the molecular basis of the apparent BZ receptor heterogeneity and the possible functional significance of BZ receptor subtypes are presented. Studies of BZ receptor heterogeneity have provided insights into the molecular events that may be responsible for BZ modulation of gamma-aminobutyric-ergic function.     

Apolipoproteins as the basis for heterogeneity in high-density lipoprotein2 and high-density lipoprotein3. Studies by isoelectric focusing on agarose films

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two-dimensional IEF-SDS electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pI in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmuno-assays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoD. ApoCII and apoCII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3.(ABSTRACT TRUNCATED AT 400 WORDS)     

脯氨酸-4-羟化酶在大肠杆菌中的密码子优化表达及对反式-4-羟脯氨酸生物合成的作用

为了使脯氨酸-4-羟化酶基因在重组大肠杆菌中得到高表达,通过调整大肠杆菌密码子偏好性以及mRNA二级结构,使得脯氨酸-4-羟化酶基因得到优化。将优化后的脯氨酸-4-羟化酶基因插入含有色氨酸串联启动子的p UC19质粒,构建重组质粒p UC19-ptrp2-Hyp,并导入大肠杆菌BL21(DE3)中。在摇瓶水平,重组菌以L-脯氨酸为底物发酵8 h,可积累(0.492±0.034)g/L的反式-4-羟脯氨酸。在发酵罐水平,通过补料分批发酵来提高反式-4-羟脯氨酸的产量,当补糖速率为18 g/h时,反式-4-羟脯氨酸的产量高达42.5 g/L,反式-4-羟脯氨酸产率为0.966 g/(L·h)。